Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed
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《核酸研究医学期刊》
Department of Functional Genomics, Exiqon, Bygstubben 9, DK-2950 Vedbaek, Denmark, 1 Department of Evolutionary Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen ?, Denmark and 2 Laboratory of Oncology 54O5, Herlev University Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark
*To whom correspondence should be addressed. Tel: +45 45 66 08 88; Fax: +45 45 66 18 88; Email: jacobsen@exiqon.com
ABSTRACT
LNA oligonucleotides constitute a class of bicyclic RNA analogues having an exceptionally high affinity for their complementary DNA and RNA target molecules. We here report a novel method for highly efficient isolation of intact poly(A)+ RNA using an LNA-substituted oligo(dT) affinity ligand, based on the increased affinity of LNA-T for complementary poly(A) tracts. Poly(A)+ RNA was isolated directly from 4 M guanidine thiocyanate-lysed Caenorhabditis elegans worm extracts as well as from lysed human K562 and vincristine-resistant K562/VCR leukemia cells using LNA_2.T oligonucleotide as an affinity probe, in which every second thymidine was substituted by LNA thymidine. In accordance with the significantly increased stability of the LNA_2.T–A duplexes in 4 M GuSCN, we obtained a 30- to 50-fold mRNA yield increase using the LNA-substituted oligo(T) affinity probe compared with DNA-oligo(dT)-selected mRNA samples. The LNA_2.T affinity probe was, furthermore, highly efficient in isolation of poly(A)+ RNA in a low salt concentration range of 50–100 mM NaCl in poly(A) binding buffer, as validated by selecting the mRNA pools from total RNA samples extracted from different Saccharomyces cerevisiae strains, followed by northern blot analysis. Finally, we demonstrated the utility of the LNA-oligo(T)-selected mRNA in quantitative real-time PCR by analysing the relative expression levels of the human mdr1 multidrug resistance gene in the two K562 cell lines employing pre-validated Taqman assays. Successful use of the NH2-modified LNA_2.T probe in isolation of human mRNA implies that the LNA-oligo(T) method could be automated for streamlined, high throughput expression profiling by real-time PCR by covalently coupling the LNA affinity probe to solid, pre-activated surfaces, such as microtiter plate wells or magnetic particles.
INTRODUCTION
Efficient selection of intact polyadenylated mRNA from eukaryotic cells and tissues is an essential step for a wide selection of functional genomics applications, including full-length cDNA library construction, EST sequencing, northern and dot-blot analyses, gene expression profiling by microarrays and quantitative real-time PCR. The key to successful selection of intact poly(A)+ RNA is fast extraction of total RNA from cells and tissues using strong denaturing agents to disrupt the cells with the simultaneous denaturation of endogenous RNases followed by mRNA sample preparation from the extracted total RNA (1–3). Chirgwin et al. (2) improved the isolation of biologically active total RNA from tissues enriched in RNases by homogenization in guanidine thiocyanate (GuSCN) and 2-mercaptoethanol followed by ethanol precipitation or by sedimentation through a caesium chloride cushion. This method was further modified by Chomczynski and Sacchi (3) to a single-step extraction of total RNA by the acid–guanidine thiocyanate–phenol–chloroform (AGPC) protocol, eliminating the ultracentrifugation step of the guanidinium–CsCl method (2). Yet another method has applied extraction with buffer-saturated phenol followed by proteinase K treatment to prevent RNA degradation (4).
Since most eukaryotic mRNAs contain tracts of poly(A) tails at their 3'-termini, polyadenylated mRNA can be selected by oligo(dT)–cellulose chromatography. Although peptide nucleic acid (PNA) analogues have recently been used for poly(A)+ RNA isolation (5), oligo(dT) continues to be the most exploited affinity ligand in mRNA sample preparation (3). More recently, a single-step poly(A)+ RNA isolation method has been described using streptavidin-coated superparamagnetic beads (6). While the direct method significantly reduces the handling and purification time, the need for a high salt concentration to stabilize the dT–A duplexes often results in co-purification of non-polyadenylated RNAs. Moreover, the poly(A) selection is carried out directly in crude cell lysates without the presence of RNase inhibitors, thereby increasing the mRNA susceptibility to RNA degradation.
Locked nucleic acid (LNA) oligonucleotides comprise a novel class of bicyclic RNA analogues having an exceptionally high affinity for their complementary DNA and RNA target molecules (7,8). We here describe a method for highly efficient isolation of intact poly(A)+ RNA from 4 M GuSCN-lysed cell extracts using a LNA-substituted oligo(dT) affinity probe, based on the increased affinity of LNA-T for complementary poly(A) tracts. In addition, we demonstrate that use of a LNA-substituted oligo(dT) probe enables efficient isolation of poly(A)+ RNA from extracted total RNA samples in a low salt binding buffer.
MATERIALS AND METHODS
Determination of the duplex melting temperatures
The melting temperatures of the LNA/RNA and the corresponding DNA/RNA duplexes were determined as described by Wahlestedt et al. (9) using 1.0 μM concentrations of the two complementary oligonucleotides in a medium salt buffer (10 mM sodium phosphate buffer, pH 7.0, 100 mM NaCl, 0.1 mM EDTA).
In vitro synthesis of polyadenylated yeast ACT1 and THI4 spike RNAs
Genomic DNA was prepared from Saccharomyces cerevisiae wild type (BY4741, MATa, his31, leu20, met150, ura30) (EUROSCARF) using the Nucleon MiY DNA extraction kit (Amersham Biosciences, UK) according to the manufacturer’s instructions. Amplification of the 3'-end of the ACT1 (YFL039C) and THI4 (YGR144W) open reading frames (ORFs) was performed by standard PCR using yeast genomic DNA as template. In the first PCR step, a forward primer containing a restriction enzyme site and a reverse primer containing a universal linker sequence were used and 20 bp was added to the 3'-end of the ORFs next to the stop codon. In the second step the reverse primer was exchanged with a nested primer containing a poly(dT)20 tail and a restriction site. The actin amplicon contained 720 bp of the ACT1 ORF plus a 20 bp universal linker sequence and a poly(dA)20 tail. The THI4 amplicon contained 723 bp of the THI4 ORF plus a 20 bp universal linker sequence and a poly(dA)20 tail. The PCR primers used were: YFL039C- Rev-Uni (5'-gatccccgggaattgccatgttagaaacacttgtggtgaacga-3') and YFL039C-For-EcoRI (5'-acgtgaattctttccatccaagccgttttg-3') for ACT1; YGR144W-Rev-Uni (5'-gatccccgggaattgccatgctaagcagcaaagtgtttcaaa-3') and YGR144W-For-EcoRI (5'-acgtgaattctcgccaagaacagaccagact-3') for THI4; Uni-polyT-BamHI (5'-acgtggatccttttttttttttttttttttgatccccgggaattgccatg-3') for both amplicons. The PCR products were digested with the restriction enzymes EcoRI and BamHI, ligated into the pTRIamp18 vector (Ambion, UK) using a Quick Ligation Kit (New England Biolabs, USA) according to the supplier’s instructions and transformed into Escherichia coli DH-5. DNA sequencing (ABI 377, Applied Biosystems, USA) was used to verify the plasmid constructs using M13 forward and reverse primers.
The yeast spike RNAs were synthesized using a MEGAscriptTM Kit (Ambion, UK) and BamHI-linearized ACT1 or THI4 plasmid clones as templates. The RNA preparations were treated with DNase I followed by purification using a RNeasy? Mini kit (Qiagen, Germany) according to the manufacturer’s instructions for in vitro RNA synthesis. The RNA quality was assessed by native agarose gel electrophoresis and the RNA concentration and purity were determined by measuring the absorbance at 260 and 280 nm.
Preparation of the magnetic particles for poly(A)+ RNA isolation
For each mRNA sample preparation, 60 μl of streptavidin-coated magnetic particles (Roche, Germany) were pipetted into an RNase-free siliconized tube (Ambion, UK). A magnetic separator (Roche, Germany) was used to remove the supernatant. An aliquot of 100 μl of 1 μg/ml yeast tRNA (Ambion, UK) diluted in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.5) was added and the mixture was incubated for 5 min at room temperature. The particles were washed in 100 μl of TE buffer and resuspended in 100 μl of 1x binding buffer . To the particles was added 200 pmol biotinylated DNA- or LNA-oligo(T) capture probe (Exiqon, Denmark) followed by incubation for 5 min at 37°C. The particles were washed twice in 1x binding buffer before collection and release to the RNA sample or lysed, cell-free sample preparation extract.
Tosyl-activated Dynabeads M-280 (Dynal, Norway) were washed in 0.1 M borate buffer, pH 9.5. To 500 μl of particles was added 10.5 nmol NH2-modified DNA- or LNA-oligo(T) capture probe , followed by incubation for 20 h at 37°C with slow end-over-end rotation. After incubation, the Dynabeads were collected with a Magnetic Particle Concentrator (MPC) (Dynal). The tube was placed in the MPC for 5 min to allow the beads to be collected by the magnet in it and the supernatant was discarded while the tube was in the MPC. The coated beads were then washed, using the MPC, twice for 10 min in 25 mM sodium citrate, 0.5% N-lauryl sarcosine and then overnight at 37°C with 500 μl of 220 mM Tris, pH 8.0, 0.05% N-lauryl sarcosine, 1 mM EDTA, pH 8.0. The coated beads were collected with the MPC, the supernatant discarded and the beads were resuspended in the GuSCN lysis buffer at a concentration of 30 mg/ml.
Yeast spike RNA recovery assays
The yeast spike RNA samples were denatured for 10 min at 65°C followed by quenching on ice for 10 min. For the GuSCN step gradient recovery assay 0.5 μg aliquots of the THI4 spike RNA were mixed in 100 μl of GuSCN lysis buffer (25 mM sodium citrate) (J.T. Baker, USA), pH 7.0, 0.5 g/100 ml sodium N-lauroyl sarcosinate) and 0, 0.5, 1, 2 or 4 M GuSCN (Sigma, USA), respectively. For capture in NaCl binding buffer, the ACT1 spike RNA samples were prepared by mixing 0.5 or 1 μg spike RNA in a final volume of 50 μl of DEPC-treated H2O (Ambion, UK) and 50 μl of 2x binding buffer , with the following NaCl concentrations in the 2x binding buffer; 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 M, respectively. All the samples were subsequently used in the poly(A)+ RNA isolation protocol with oligo(dT) and LNA-oligo(T) capture. The magnetic particles were transferred to the spike RNA mixture and incubated for 10 min at 37°C in an Eppendorf Thermomixer at 400 r.p.m. (Eppendorf, Germany). The particles were collected and released into the wash buffer , re-collected and the washing step was repeated twice. Finally, the poly(A)+ RNA was eluted in 10 μl of DEPC-treated H2O by heating the sample at 65°C for 10 min, quenching on ice and removing the particles. For subsequent analysis, 3 μl of the sample and a standard dilution series of either THI4 or ACT1 were applied on a 1% native agarose gel. The gel was subjected to electrophoresis for 20–30 min at 7 V/cm and then quantified on a Typhoon 9200 Imager (Amersham Biosciences, USA).
Yeast cultures and extraction of yeast total RNA
Saccharomyces cerevisiae wild type (BY4741, MATa, his31, leu20, met150, ura30) and a hsp78 mutant strain (BY4741, MATa, his31, leu20, met150, ura30, YDR258c::kanMX4) (EUROSCARF) were grown in YPD medium at 30°C until the A600 of the cultures reached 0.8. Half of the cultures were collected by centrifugation and resuspended in 1 vol of 40°C pre-heated YPD. Incubation was continued for an additional 30 min at 30 or 40°C for the standard and heat-shocked cultures, respectively. Cells were harvested by centrifugation and stored at –80°C. Yeast total RNA was extracted using a FastRNA Kit-RED (BIO 101, USA) according to the manufacturer’s instructions. The quantity and quality of the total RNA preparations were assessed by standard spectrophotometry or using a NanoDrop ND-1000 (NanoDrop, USA) combined with agarose gel electrophoresis.
Isolation of yeast poly(A)+ RNA
An aliquot of 100 μg yeast total RNA was combined in a final volume of 50 μl of 1x binding buffer and heated for 30 min at 65°C, 700 r.p.m. The tube was spun briefly and quenched on ice for 10 min. To the total RNA sample was added 200 pmol biotinylated DNA-oligo(dT)20 or LNA_2.T capture probe and the samples were incubated for 15 min at 37°C, followed by addition of streptavidin-coated particles and incubation of the sample preparation for 10 min at 37°C, 400 r.p.m. The magnetic particles were collected and released into the wash buffer, re-collected and the washing step was repeated twice. Finally, the poly(A)+ RNA was eluted in 10 μl of DEPC-treated H2O by heating the sample at 65°C for 10 min, quenching on ice and removing the particles.
Caenorhabditis elegans cultures
The C.elegans N2 Bristol strain was grown in S-medium, with E.coli NA22 food, at 23°C and was cleaned by standard sucrose density methods as described (10). Caenorhabditis elegans mixed stage worms were harvested and resuspended in 4 vol of GuSCN lysis buffer , immediately frozen in liquid nitrogen and stored at –80°C.
Isolation of C.elegans poly(A)+ RNA from 4 M GuSCN-lysed worm extracts
Caenorhabditis elegans samples stored in the GuSCN lysis buffer (see above) were thawed and the wet weight was calculated by removing the supernatant (centrifugation for 2 min at 4000 g) and weighing. The pellet was subsequently resuspended in the same volume. The C.elegans mixed stage worm aliquots were spun for 2 min at 4000 g and mixed with 200 μl of GuSCN lysis buffer. Quartz sand was added to the C.elegans samples and the samples were disrupted for 2 min on ice using a pestle. The extract was heated for 30 min at 65°C, 700 r.p.m., incubated on ice for 10 min and briefly spun down. The soluble, 4 M GuSCN-lysed C.elegans extract was transferred to a clean, RNase-free tube containing the LNA_2.T or DNA-oligo(dT)20 capture probe coupled to magnetic particles and incubated for 5 min at 37°C, 400 r.p.m. for poly(A)+ RNA selection. The particles were re-collected and released into the wash buffer and the washing step was repeated twice. Finally, the poly(A)+ RNA was eluted in 50 μl of DEPC-treated H2O. The samples were incubated for 10 min at 65°C and quenched on ice for 10 min to release the polyadenylated RNA from the particles. The particles were removed and the supernatant was spun briefly (1 min at 16 100 g) and transferred to a clean RNase-free siliconized tube. The isolated poly(A)+ RNA was ethanol precipitated by adding 0.1 vol 3 M NaOAc (Ambion, UK), 150 μg/ml Glycogen Carrier (Ambion, UK) and 2.5 vol 96% ethanol to the tube and stored at –20°C overnight. After spinning for 30 min at 16 100 g at 4°C the supernatant was removed and the pellet washed with ice-cold 70% ethanol and air dried. The pellet was dissolved in 10 μl of DEPC-treated H2O.
Isolation of poly(A)+ RNA from human K562 and vincristine-resistant K562/VCR cells
The K562 leukemia cells (a human erythroleukemia cell line derived from a chronic myeloid leukemia patient in blast crisis) were cultured in HEPES-buffered RPMI 1640 medium containing 5% newborn calf serum, 1% L-glutamine, 100 U penicillin and 100 mg/ml streptomycin (all from Gibco BRL Life Technologies, Denmark). All cultures were maintained at 37°C in an atmosphere of 95% air and 5% CO2. The resistant cells were kept in sub-lethal concentrations of drug. Wild-type K562 cells were used to develop the vincristine-resistant cell line. The cells were cultured at increasing drug concentrations for 35 passages. The drug resistance levels were measured by a clonogenic assay (11). In brief, single cell suspensions with the desired drug concentration were plated in soft agar containing sheep red blood cells and 2-mercaptoethanol. The plates were exposed continuously to the drugs and the experiments were performed in triplicate. The IC50 values were defined as the concentrations inhibiting 50% of colony formation, while the relative resistance was defined as the ratio between the IC50 value for the resistant line and the wild-type cell line. The K562/VCR cell line is 152 times more resistant to the chemotherapeutic drug vincristine compared to drug-sensitive K562. The cell cultures were harvested by centrifugation, the supernatant was removed and the pellets were frozen in N2 and stored at –80°C until use. The pellets were subsequently resuspended in the 4 M GuSCN lysis buffer containing 10 mM dithiothreitol (DTT) (107 cells/100 μl). Quartz sand was added and the samples were treated as described for the worm extracts. The human cell extracts were diluted to 106 cells/50 μl in the GuSCN lysis buffer containing 10 mM DTT before incubating with the LNA_2.T or DNA-oligo(dT)20 capture probe coupled to magnetic particles (5 min at 37°C) for poly(A)+ RNA selection. The particles were re-collected and released into wash buffer , the washing step being repeated twice. The last two washing steps were without sodium N-lauryl sarcosinate. Finally, the poly(A)+ RNA was eluted twice in 10 μl of DEPC-treated H2O by incubating for 10 min at 65°C and quenched on ice for 10 min to release the polyadenylated RNA from the particles. The eluates were pooled and treated as described above.
RT–PCR and quantitative real-time RT–PCR assays
An aliquot of 100 ng poly(A)+ RNA from mixed stage C.elegans worms was combined with 5 μg oligo(dT)12–18 primer (Amersham Biosciences, UK) and heated for 10 min at 70°C, followed by quenching on ice. First strand cDNA was synthesized by transferring the mixture to 20 μl of cDNA synthesis reaction containing 50 mM Tris–HCl (pH 8.3 at room temperature), 75 mM KCl, 3 mM MgCl2, 10 mM DTT (Invitrogen, USA), 1 mM each dATP, dCTP, dGTP and dTTP (Amersham Biosciences, USA) and 20 U Superasin (Ambion, UK) and incubating for 5 min at 37°C. An aliquot of 200 U SuperScriptTM II RT (Invitrogen, USA) was added and the reaction mixture was incubated for 30 min at 37°C and 30 min at 42°C. An additional 200 U SuperScriptTM II RT was added and the incubation at 42°C was prolonged for 1 h. Finally, the reaction was diluted five times in DEPC-treated H2O and heated for 5 min at 70°C and the oligo(dT)12–18 primer was removed on a Sephacryl S-400 HR spin column (Amersham Biosciences, UK) according to the manufacturer’s instructions. First strand cDNA was generated from poly(A)+ RNA isolated from human K562 and K562/VCR cells essentially as described above, except that 5 μg random hexamers were used instead of the oligo(dT)12–18 primer. The excess primer was removed on a Microcon YM-30 filter unit (Millipore, USA).
Four PCR fragments were amplified from first strand worm cDNA using gene-specific primer sets for: (i) C.elegans 26S rRNA (GenBank accession no. Z92784 ), 5'-gccagaggaaactctggtggaagtcc-3' and 5'-agcctcccttggtgttttaagggccg-3'; (ii) ?-actin (GenBank accession no. Z71181 ), 5'-ggattcgcaagggcgaaaggtgattg-3' and 5'-gctttattccaagtttggccatac-3'; (iii) let-2 (GenBank accession no. AAA96216 , 5'-gatcgaattcctccaggagagaagggagatg-3' and 5'-gatcaagcttatctctt cctgggtatccagctt-3'; (iv) T01D3.3 (GenBank accession no. Z81110 ), 5'-gatcgaattcatgatacctgcagatgtgcctgcctac-3' and 5'-gatcgagctccaatggatgttcacagtgttgctcg-3'. PCR reactions (25 μl) were carried out by mixing 15 mM Tris–HCl, pH 8.0, 50 mM KCl (GeneAmp Gold buffer; PE Biosystems, USA), 2.5 mM MgCl2, 200 μM each dATP, dCTP, dGTP and dTTP, 0.4 mmol/l forward primer; 0.4 mM reverse primer; 1.25 U (0.25 μl of a 5 U/μl solution) AmpliTaq Gold polymerase (PE Biosystems, USA) and 1 μl of cDNA as template. After an initial 5 min denaturation step at 95°C, 30 cycles of PCR were carried out (45 s at 95°C, 30 s at 60°C and 90 s at 72°C), followed by extension at 72°C for 10 min. The PCR products were analysed by native agarose gel electrophoresis. The 26S rRNA PCR fragment was purified by agarose gel electrophoresis followed by a Qiaquick PCR purification kit (Qiagen, Germany) for probe labelling according to the supplier’s instructions. Two PCR fragments were amplified from first strand human K562 cell line cDNA using gene-specific primer sets for: (i) human mdr1 (GenBank accession no. NM_000927 ), 5'-ttctagcccttggaattatttcttt-3' and 5'-ctgatattttggctttggcata-3'; (ii) ?-actin (GenBank accession no. NM_001101 ), 5'-tgccctgaggcactcttc-3' and 5'-cgatccacacggagtacttg-3'. The PCR reactions and cycling conditions were as described above.
Quantitative real-time PCR assays for the human mdr1 and ?-actin mRNAs were performed using 1x TaqMan Universal PCR Master Mix, No AmpErase UNG and 1x TaqMan Assays-on-Demand, Gene Expression Product for the mdr1 mRNAs (Assay ID Hs00184491_m1) or 1x TaqMan Pre-Developed Assay Reagent endogenous control for the ?-actin mRNA (TaqMan PDAR human ACTB, catalogue no. 4310881E) in an ABI PRISM 7000 Sequence Detection System as follows: 10 min denaturation at 95°C, followed by 50 cycles of denaturation for 15 s at 95°C and annealing and elongation for 1 min at 60°C using 2 μl of the diluted cDNA reaction from above as template in a final volume of 25 μl. The relative gene expression level of the mdr1 mRNA in the two human leukemia cell lines was calculated as described in User Bulletin no. 2 for the ABI PRISM 7700 Sequence Detection System. The expression data obtained represent average values from a minimum of three replicate experiments. All samples were normalized using the TaqMan PDAR human ACTB endogenous control for detection of the ?-actin mRNA level treated as an internal control. Unless otherwise stated, all reagents were purchased from Applied Biosystems (USA).
Northern blot analysis
Aliquots (500 ng/sample) of the LNA oligo_2.T-selected poly(A)+ RNA samples from wild type yeast cells and heat shocked wild type and hsp78 mutant cells, respectively, were subjected to electrophoresis in a 1.5% agarose–2.2 M formaldehyde gel (12) and blotted onto Hybond-N nylon membrane (Amersham Biosciences, UK) with 10x SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.0) as transfer buffer (13). A 756 bp PCR amplicon of the yeast HSP78 gene was 32P-labelled (>5 x 108 c.p.m./μg) by random priming (MegaprimeTM DNA labelling system; Amersham Biosciences, UK) and purified according to the manufacturer’s recommendations. Redivue dCTP (3000 Ci/mmol) was purchased from Amersham Biosciences (UK). The radioactively labelled probe was hybridized to the filter at 42°C for 18 h in ULTRA-hybTM (Ambion, UK). The filter was washed twice in 2x SSC, 0.1% SDS at 42°C for 5 min, then twice in 0.1x SSC, 0.1% SDS at 42°C for 15 min. After autoradiography on a Storage Phosphor screen (Amersham Biosciences, UK) and image analysis quantification using a Typhoon 9200 scanner (Amersham Biosciences, UK), the probe was removed from the filter according to the manufacturer’s instructions. The filter was rehybridized with a 748 bp PCR amplicon of the yeast ACT1 gene. 32P-labelling of the probe, hybridization to the filter and the washing steps were identical to those described for the HSP78 probe.
Aliquots of the LNA-oligo_2.T and DNA-oligo(dT)20 selected worm poly(A)+ RNA samples (4 μl/sample, 2.8, 5.5, 11, 22 and 44 mg worm extract, respectively) together with 10 μg C.elegans total RNA from worm embryos were subjected to northern blot analysis as described above. A 483 bp PCR amplicon of the RPL-21 cDNA (GenBank accession no. AAA27951 (a gift from M. Zagrobelny, University of Copenhagen, Denmark) was 32P-labelled (>5 x 108 c.p.m./μg). Hybridization to the filter and the washing steps were performed as described above. The filter was sequentially rehybridized with the let-2, mouse GAPDH (Ambion, USA) and 989 bp 26S rDNA fragments. 32P-labelling of the probes, hybridization to the filter and the washing steps were identical to those with the RPL-21 probe.
Poly(A)+ RNA aliquots of the LNA_2.T and DNA-oligo(dT)20 selected human K562 and K562/VCR samples were subjected to northern blot analysis as described above, except that the agarose gel was 1%. The mouse GAPDH (Ambion, USA) cDNA fragment was 32P-labelled and hybridized to the filter as described above.
RESULTS AND DISCUSSION
Total, cellular RNA is typically extracted from cells and tissues using strong chaotropic agents, which lyse the cells with simultaneous denaturation of endogenous RNases (2,3), followed by poly(A)+ RNA selection from the purified total RNA (1). The chaotropic salt GuSCN is widely used to lyse cells and release nucleic acids into solution due to its ability to effectively denature secondary/tertiary protein and nucleic acid structures (14). In addition to efficient cell lysis, its use in extraction buffers at a high concentration thus also leads to concomitant inhibition of endogenous proteases and nucleases, including RNases (2,15). In order to be effective both as a chaotrope and an RNase inhibitor, GuSCN has to be used at a high concentration of at least 4 M (16). In turn, this dramatically decreases the thermal stability of oligonucleotide duplexes, including dT–A duplexes exploited when using oligo(dT) affinity chromatography in poly(A) selection (17). Consequently, DNA-oligo(dT) chromatography has to date only been demonstrated at low concentrations of GuSCN, which is not sufficient for RNase inhibition (18,19).
To evaluate the hybridization properties of LNA-substituted oligo(dT) affinity probes, different LNA-oligo(T) oligonucleotides were designed and synthesized (Table 1). The duplex thermal stabilities of the different dT–A duplexes with complementary RNA were determined by measuring the melting temperatures (Tm). Substitution of a DNA-oligo(dT)20 oligonucleotide by LNA-T resulted in significantly increased thermal duplex stabilities in all LNA-oligo(T) designs measured, corresponding to an increase in melting temperature ranging from +2.8 to +6.0°C per LNA thymidine monomer. This is in good agreement with previous results reported for extensively modified oligothymidylate LNA oligonucleotides (20). Among the LNA-T probes, the fully substituted LNA-T20 and LNA-T15 oligonucleotides showed a Tm of >95°C, indicating that their exceptionally high thermal stability would not allow efficient elution of the captured poly(A)+ RNA from the affinity ligand. By comparison, LNA_2.T, in which every second thymidine was substituted with LNA-T, showed a Tm of 70.8°C and an increase of 30°C compared to the DNA control probe. Thus, the LNA_2.T affinity probe represented an adequate compromise between increased duplex thermal stability and melting of the dA–T duplexes in elution buffer. In our initial experiments the efficiency of all the different LNA-T-substituted affinity probes (Table 1) in poly(A)+ RNA isolation was assessed along with the reference DNA-oligo(dT)20 probe using polyadenylated, in vitro synthesized yeast spike RNAs. The recovery of the spike RNAs from GuSCN-containing binding buffer was consistently highest using the LNA_2.T affinity probe (data not shown). Thus, the LNA-oligo_2.T probe was chosen for all further experiments.
Table 1. Thermal duplex stabilities of different LNA-substituted oligo(T) affinity probes
The recovery of polyadenylated yeast THI4 spike RNA directly from a chaotropic lysis buffer was assessed using either the LNA_2.T or the reference DNA-oligo(dT)20 probe by increasing the final GuSCN concentration stepwise from 0 to 4 M GuSCN (Fig. 1A). The spike RNA recovery was low with the DNA control over the whole GuSCN concentration range (0.5–4 M) (Fig. 1A). The optimal GuSCN concentration for the reference DNA-oligo(dT)20 probe was found to be 0.5 M GuSCN (20% spike RNA recovery), in accordance with previous results reported with oligo(dT) chromatography (18). In contrast, the THI4 spike RNA recovery with the LNA_2.T probe was unaffected by increasing the GuSCN concentration in the binding buffer, showing comparable yields of 80% over the entire range from 0.5 to 4 M GuSCN. The results thus implied that the LNA_2.T affinity probe would be useful in direct isolation of poly(A)+ RNA from cell or tissue extracts lysed in 4 M GuSCN buffer, which effectively inhibits endogenous RNases.
Figure 1. The impact of (A) GuSCN and (B) NaCl concentration on the recovery of yeast spike RNAs. (A) Aliquots (500 ng) of the 0.8 kb yeast THI4 spike RNA were captured at increasing final GuSCN concentrations in binding buffer from 0 to 4 M GuSCN using the reference DNA-oligo(dT)20 and LNA_2.T affinity probes, electrophoresed in a 1% agarose gel stained with Gelstar. Lanes 1–6, THI4 spike RNA standard curve (STD) with 250, 125, 62.5, 31.3, 15.6 and 7.8 ng/lane, respectively; lanes 7–11, THI4 spike RNA captured by the reference DNA-oligo(dT)20 in 0, 0.5, 1, 2 and 4 M GuSCN, respectively; lanes 12–16, THI4 spike RNA captured by the LNA_2.T affinity probe in 0, 0.5, 1, 2 and 4 M GuSCN, respectively. A 1 kb DNA ladder (Invitrogen, USA) was used as a size marker. Recovery (%) of the yeast THI4 RNA by the reference DNA-oligo(dT)20 (open bars) and the LNA_2.T (solid bars) affinity probes, respectively, at increasing GuSCN concentrations was calculated by image analysis of the agarose gel. (B) Aliquots (1 μg) of the 0.8 kb yeast ACT1 spike RNA were captured at increasing final salt concentrations in binding buffer from 0 to 0.5 M NaCl using the reference DNA-oligo(dT)20 and LNA_2.T affinity probes and electrophoresed in a 1% agarose gel stained with Gelstar (not shown). Recovery (%) of the yeast ACT1 RNA by the reference DNA-oligo(dT)20 (open bars) and the LNA_2.T (solid bars) affinity probes, respectively, at different NaCl concentrations (0–0.5 M) was calculated by image analysis of the agarose gel.
Next, we tested the same oligo(T) affinity probes in a spike RNA recovery assay employing different hybridization conditions in a non-chaotropic poly(A) binding buffer by varying the salt concentration range from 0 to 0.5 M NaCl (Fig. 1B). A high spike RNA recovery of 70–100% was observed for both the reference DNA-oligo(dT)20 and LNA_2.T affinity probes in the high salt concentration range 0.2–0.5 M NaCl in the binding buffer (Fig. 1B). However, in the low salt range 50–100 mM a significantly decreased recovery of the ACT1 spike RNA was observed with the reference DNA-oligo(dT)20 probe, while the recovery was still between 80 and 90% with the LNA-oligo(T) affinity ligand, indicating that a low salt, high hybridization stringency window could be employed in combination with the LNA-oligo(T) affinity probe without compromising the mRNA yield.
The validity of this conclusion was assessed by poly(A)+ RNA selection experiments with yeast RNA samples. Total RNA was extracted by standard methods from S.cerevisiae wild type, heat-shocked wild type and heat-shocked hsp78 mutant cells, respectively. The hsp78 mutant is a strain in which the HSP78 gene is deleted and thus HSP78 mRNA is not present in these cells (21). Yeast poly(A)+ RNA was subsequently isolated from the total RNA samples using the LNA_2.T affinity probe under low salt (100 mM) binding conditions and the quality was assessed by northern blot analysis (Fig. 2). The mRNA yield in the LNA_2.T-selected poly(A)+ RNA samples corresponded to a recovery of 2.6–3.8% of the input yeast total RNA. The hybridized northern blot revealed a prominent 2.3 kb message in the heat-shocked wild-type strain and a weakly hybridizing HSP78 mRNA in the wild-type control (Fig. 2). Furthermore, the HSP78 mRNA was 14-fold up-regulated as a response to the heat shock treatment in the wild type, whereas no HSP78 transcript was detected from the heat-shocked hsp78 mutant strain mRNA sample, as expected. By comparison, the ACT1, which was used as a loading control in the northern analysis, revealed a 1.1 kb mRNA of comparable intensity in all three yeast samples (Fig. 2). All the isolated yeast mRNA samples were fully intact as judged by the northern blot analysis. In conclusion, the LNA_2.T affinity probe is highly efficient in isolation of poly(A)+ RNA at a low salt concentration of 100 mM NaCl in the poly(A) binding buffer. We therefore anticipate that low salt, high stringency hybridization conditions would be highly preferable compared to the standard method employing 0.5 M NaCl in the binding buffer, due to the enhanced destabilization of secondary structures in the mRNA.
Figure 2. Northern blot analysis of yeast poly(A)+ RNA isolated by the LNA_2.T affinity probe under low salt binding conditions. Yeast poly(A)+ RNA was isolated from wild type, wild type heat shocked and hsp78 mutant heat shocked total RNA and probed sequentially with 32P-labelled fragments for the yeast HSP78 and ACT1 genes, respectively.
To assess whether the LNA-oligo(T) capture method would allow mRNA sample preparation directly from cell or tissue extracts lysed in 4 M GuSCN buffer, we isolated poly(A)+ RNAs from C.elegans worm extracts using the LNA_2.T affinity probe in comparison with the reference DNA-oligo(dT)20 probe. The isolated worm poly(A)+ RNA samples were subsequently evaluated by northern blot analysis (Fig. 3). The cDNA probes for the C.elegans let-2, ?-actin and RPL-21 mRNAs readily detected single mRNA species in the worm samples selected with the LNA_2.T affinity probe, whereas the samples captured with the reference DNA-oligo(dT)20 probe showed only weakly hybridizing fragments on the northern blot (Fig. 3). Moreover, the message levels for all three transcripts increased as a function of increased worm extract sample size in the LNA_2.T selected samples, indicating that poly(A) selection by the LNA-substituted affinity probe is specific for polyadenylated mRNAs. This is in good agreement with the results obtained with the yeast spike RNA recovery assay (Fig. 1A) and implies that the reference DNA-oligo(dT)20 probe is unable to form stable dT–A duplexes in the presence of 4 M GuSCN. None of the isolated worm mRNA samples showed degradation by RNases, as judged by the northern analysis. Re-probing the same northern filter with a 32P-labelled DNA fragment for the C.elegans 26S rRNA probe revealed a prevalent, strongly hybridizing 3.5 kb rRNA species in the control total RNA sample, corresponding to the 26S rRNA, while only weakly hybridizing 26S rRNA fragments were detected in the poly(A) selected worm mRNA samples (Fig. 3). Estimation of the mRNA levels based on the data obtained by northern blot image analysis for the three C.elegans transcripts (Fig. 3) implies that the mRNA yield by the LNA_2.T affinity probe is more than 50-fold higher compared to the DNA control probe, when using the same worm extract sample size. On the other hand, the ratios of the let-2, GAPDH and RPL-21 mRNAs to the 26S rRNA were estimated to be 1.3, 35 and 191, respectively, for the LNA-oligo(T)-selected mRNA samples compared to those of the DNA-oligo(dT)20 control samples, where the ratios were estimated to be 0.1, 7 and 17 thus further supporting the notion that LNA_2.T is highly specific for polyadenylated mRNAs.
Figure 3. Direct isolation of poly(A)+ RNA from 4 M GuSCN-lysed C.elegans worm extracts using LNA-oligo(T) capture. Northern blot analysis of the poly(A)+ RNA samples from 4 M GuSCN-lysed mixed stage C.elegans worm extracts is shown as a function of increasing sample size (2.8, 5.5, 11, 22 and 44 mg wet weight), selected using the DNA-oligo(dT)20 or the LNA_2.T affinity probe. The same filter was sequentially hybridized with 32P-labelled DNA fragments for the C.elegans RPL-21, let-2 and GAPDH mRNAs and cytosolic 26S rRNA. Ten micrograms of total RNA isolated from C.elegans embryos was used as a control.
Next, we subjected the isolated worm poly(A)+ RNA samples to reverse transcription (RT) PCR assays for C.elegans mRNAs representing three abundance classes, in accord with previously published, compiled data from genome-wide expression profiling in C.elegans, reporting the following expression levels from embryos to adult worms for the aforementioned three messages; 35–207 p.p.m. for let-2, 6–21 p.p.m. for ?-actin and 3–8 p.p.m. for T01D3.3 (22). The LNA_2.T- and reference DNA-oligo(dT)20-captured poly(A)+ RNA samples were reversed transcribed to first strand cDNA using standard methods followed by PCR amplification of cDNA fragments for C.elegans ?-actin, let-2 and T01D3.3 (23), respectively. Analysis of the PCR products obtained by RT–PCR of LNA_2.T-selected mRNA revealed single, prominent PCR amplicons of the expected size with each of the three primer pairs (data not shown). By contrast, only a weak PCR amplicon for the let-2 cDNA was detected by RT–PCR of the DNA-oligo(dT)20-selected control worm mRNA, in accordance with the low yield at 4 M GuSCN concentration.
In order to apply the LNA-oligo(T) capture method to mRNA sample preparation from human cells, we isolated poly(A)+ RNAs directly from 4 M GuSCN-lysed K562 and K562/VCR leukemia cells, respectively, using the biotinylated LNA_2.T probe along with a 5' NH2-modified LNA_2.T probe, which allows covalent coupling of the affinity probe onto pre-activated magnetic particles. The yield was 300 ng poly(A)+ RNA from 106 K562 cells with both LNA probes, whereas no mRNA could be captured with the DNA-oligo(dT)20 control probes, in accord with our previous results. Northern blot analysis of the poly(A)+ RNA samples revealed a single 1.3 kb mRNA species for the human GAPDH gene in the K562 and K562/VCR sample preparations selected with both LNA_2.T affinity probes (Fig. 4A).
Figure 4. Analysis of poly(A)+ RNA isolated directly from 4 M GuSCN-lysed human K562 and K562/VCR erythroleukemia cells by LNA-oligo(T) capture. (A) Northern blot analysis of the poly(A)+ RNA samples selected from 4 M GuSCN-lysed human K562 (1) and K562/VCR (2) cells, respectively, using the 5'-biotinylated or 5'-NH2-modified LNA_2.T affinity probe and the corresponding DNA-oligo(dT)20 control probes. The filter was hybridized with a 32P-labelled DNA fragment for mouse GAPDH mRNA. (B) Approximately 100 ng poly(A)+ RNA purified from the human K562 (1) and K562/VCR (2) cell lines was used as template for RT–PCR assays for human mdr1 and ?-actin. The amplicon sizes were 256 and 738 bp for the ?-actin and mdr1 mRNAs, respectively. The RT–PCR products were electrophoresed in a 1% native agarose gel and visualized by staining with Gelstar. A negative PCR control without template was performed for each assay. (C) Representative amplification plots of quantitative real-time RT–PCR assays for the human mdr1 transcript using mRNA samples isolated from human erythroleukemia cells as template. The poly(A)+ RNAs were selected either using the biotinylated LNA_2.T affinity probe from K562 cells (open triangle) and K562/VCR cells (solid triangle) or the 5'-NH2-modifed LNA_2.T affinity probe from K562 cells (open square) and K562/VCR cells (solid square). The plots relate the PCR cycle number to the change in detected, baseline-corrected fluorescence (Rn). The small, solid circles depict the fluorescence generated from the no template control reaction.
Subsequent RT–PCR assays for the human ?-actin mRNA revealed single cDNA fragments of the expected size in all four LNA_2.T-selected mRNA templates, whereas no PCR products were detected after 30 cycles of amplification from the DNA-oligo(dT)20-selected control samples (Fig. 4B). In contrast, RT–PCR for the human multidrug resistance gene mdr1 generated the 738 bp PCR amplicon in the K562/VCR cell line, but not in the drug-sensitive K562 cell line, implying that the mdr1 gene is overexpressed in K562/VCR cells, presumably reflecting their significantly increased resistance to the chemotherapeutic drug vincristine. We therefore decided to assess relative expression of the human mdr1 gene in the two K562 cell lines by quantitative real-time RT–PCR using a pre-validated TaqMan assay and ?-actin as an internal control. First, we used aliquots of the isolated poly(A)+ RNA samples as templates for reverse transcription, followed by a 5-fold dilution of the cDNA synthesis reaction prior to quantitative real-time PCR. Subsequent real-time PCR assays revealed an average increase of four orders of magnitude in mdr1 expression relative to ?-actin mRNA in the vincristine-resistant K562/VCR cell line compared to the sensitive K562 cells (Table 2 and Fig. 4C), which is in good agreement with the standard RT–PCR results. By comparison, Fujimaki et al. (24) have reported relative mdr1 expression levels of 0.21–6.7 in different K562/VCR cell lines, whereas the level was <0.0041 in other drug-sensitive cell lines, as assayed by real-time RT–PCR using GAPDH as an internal control. Our expression data were highly comparable with poly(A)+ RNA obtained with both LNA_2.T affinity capture methods (Fig. 4C). Given the average yield of 300 ng per 106 cells and the 5-fold dilution of the cDNA synthesis reaction, a single LNA-oligo(T) sample preparation would allow quantification of 33 different mRNAs in triplicate using real-time PCR assays. The fact that we were successful in substituting the biotinylated LNA_2.T affinity probe by the NH2-modified LNA_2.T probe strongly suggests that the LNA-oligo(T) method is amenable to automation for streamlined, high throughput expression profiling by real-time PCR by covalently coupling the probe to solid, pre-activated surfaces, such as microtitre plate wells or magnetic particles.
Table 2. Relative expression of the multidrug resistance gene mdr1 in human vincristine-resistant K652/VCR cells compared to drug-sensitive K562 cells
The results presented here confirm earlier reports (18) that optimal hybridization conditions for DNA-oligo(dT)20 in a GuSCN-containing lysis buffer is between 0.5 and 1 M GuSCN, whereas capture is highly inefficient at higher GuSCN concentrations. Hence, several poly(A)+ RNA isolation procedures initiate with crude cell lysates homogenized in the presence of 4 M GuSCN (25), followed by dilution of the GuSCN lysis buffer prior to poly(A)+ RNA selection by oligo(dT) capture. Thus, the existing DNA-oligo(dT) and PNA-T (5) methods have the disadvantage of selecting the poly(A)+ RNA under moderate chaotropic salt concentration where RNase inhibition could be inadequate. Furthermore, handling of many sample preparations simultaneously, e.g. in high throughput expression analyses, becomes more labour intensive and time consuming due to the additional dilution step. In contrast, the results presented in this study demonstrate that the LNA-substituted LNA_2.T affinity probe is capable of selecting poly(A)+ RNA in the presence of 4 M GuSCN in the cell lysis buffer due to its exceptionally high affinity for complementary poly(A) tracts. This enables direct and fast poly(A)+ RNA capture under conditions allowing complete inhibition of endogenous RNase activity, resulting in highly intact mRNA samples. We were successful in exploiting the biotin–streptavidin coupling chemistry in our mRNA isolation procedure by limiting the hybridization time to 5 min, in order to prevent streptavidin denaturation even in the presence of 4 M GuSCN. This is in accord with previous studies reporting that streptavidin is highly resistant to denaturation by guanidine hydrochloride (26–28). Furthermore, we demonstrated the utility of the LNA-oligo(T) sample preparation method employing a 5' NH2-modified LNA_2.T affinity probe coupled covalently to pre-activated magnetic particles, thus overcoming the potential problem of denaturation by GuSCN.
In summary, we have demonstrated that the use of the LNA affinity probe LNA_2.T, in which every second thymidine residue is substituted with a LNA-T, enables efficient isolation of poly(A)+ RNA from 4 M GuSCN-lysed cell extracts as well as from total RNA in a low salt binding buffer, due to the exceptionally high affinity of LNA_2.T for complementary poly(A) tracts. On the other hand, the similar electrostatic properties of LNA compared to DNA and RNA oligonucleotides contribute to good aqueous solubility, facilitating use of the LNA_2.T affinity probe in mRNA sample preparation as part of an automated set-up. Thus, the results described here strongly suggest that the LNA-oligo(T)-based poly(A) selection method represents an attractive alternative to current mRNA sample preparation protocols, resulting in the recovery of highly enriched and intact poly(A)+ RNA, suitable for a wide range of downstream applications, including high throughput gene expression profiling by real-time quantitative PCR.
ACKNOWLEDGEMENTS
We thank Marianne S?s Ludvigsen, Marianne Bonde Mogensen and Mette Bj?rn at Exiqon for expert technical assistance. Also Pia Friis at the Department of Evolutionary Biology, University of Copenhagen and Marianne Fregil at the Laboratory of Oncology 54O5, Herlev University Hospital are thanked for their skilled technical support.
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*To whom correspondence should be addressed. Tel: +45 45 66 08 88; Fax: +45 45 66 18 88; Email: jacobsen@exiqon.com
ABSTRACT
LNA oligonucleotides constitute a class of bicyclic RNA analogues having an exceptionally high affinity for their complementary DNA and RNA target molecules. We here report a novel method for highly efficient isolation of intact poly(A)+ RNA using an LNA-substituted oligo(dT) affinity ligand, based on the increased affinity of LNA-T for complementary poly(A) tracts. Poly(A)+ RNA was isolated directly from 4 M guanidine thiocyanate-lysed Caenorhabditis elegans worm extracts as well as from lysed human K562 and vincristine-resistant K562/VCR leukemia cells using LNA_2.T oligonucleotide as an affinity probe, in which every second thymidine was substituted by LNA thymidine. In accordance with the significantly increased stability of the LNA_2.T–A duplexes in 4 M GuSCN, we obtained a 30- to 50-fold mRNA yield increase using the LNA-substituted oligo(T) affinity probe compared with DNA-oligo(dT)-selected mRNA samples. The LNA_2.T affinity probe was, furthermore, highly efficient in isolation of poly(A)+ RNA in a low salt concentration range of 50–100 mM NaCl in poly(A) binding buffer, as validated by selecting the mRNA pools from total RNA samples extracted from different Saccharomyces cerevisiae strains, followed by northern blot analysis. Finally, we demonstrated the utility of the LNA-oligo(T)-selected mRNA in quantitative real-time PCR by analysing the relative expression levels of the human mdr1 multidrug resistance gene in the two K562 cell lines employing pre-validated Taqman assays. Successful use of the NH2-modified LNA_2.T probe in isolation of human mRNA implies that the LNA-oligo(T) method could be automated for streamlined, high throughput expression profiling by real-time PCR by covalently coupling the LNA affinity probe to solid, pre-activated surfaces, such as microtiter plate wells or magnetic particles.
INTRODUCTION
Efficient selection of intact polyadenylated mRNA from eukaryotic cells and tissues is an essential step for a wide selection of functional genomics applications, including full-length cDNA library construction, EST sequencing, northern and dot-blot analyses, gene expression profiling by microarrays and quantitative real-time PCR. The key to successful selection of intact poly(A)+ RNA is fast extraction of total RNA from cells and tissues using strong denaturing agents to disrupt the cells with the simultaneous denaturation of endogenous RNases followed by mRNA sample preparation from the extracted total RNA (1–3). Chirgwin et al. (2) improved the isolation of biologically active total RNA from tissues enriched in RNases by homogenization in guanidine thiocyanate (GuSCN) and 2-mercaptoethanol followed by ethanol precipitation or by sedimentation through a caesium chloride cushion. This method was further modified by Chomczynski and Sacchi (3) to a single-step extraction of total RNA by the acid–guanidine thiocyanate–phenol–chloroform (AGPC) protocol, eliminating the ultracentrifugation step of the guanidinium–CsCl method (2). Yet another method has applied extraction with buffer-saturated phenol followed by proteinase K treatment to prevent RNA degradation (4).
Since most eukaryotic mRNAs contain tracts of poly(A) tails at their 3'-termini, polyadenylated mRNA can be selected by oligo(dT)–cellulose chromatography. Although peptide nucleic acid (PNA) analogues have recently been used for poly(A)+ RNA isolation (5), oligo(dT) continues to be the most exploited affinity ligand in mRNA sample preparation (3). More recently, a single-step poly(A)+ RNA isolation method has been described using streptavidin-coated superparamagnetic beads (6). While the direct method significantly reduces the handling and purification time, the need for a high salt concentration to stabilize the dT–A duplexes often results in co-purification of non-polyadenylated RNAs. Moreover, the poly(A) selection is carried out directly in crude cell lysates without the presence of RNase inhibitors, thereby increasing the mRNA susceptibility to RNA degradation.
Locked nucleic acid (LNA) oligonucleotides comprise a novel class of bicyclic RNA analogues having an exceptionally high affinity for their complementary DNA and RNA target molecules (7,8). We here describe a method for highly efficient isolation of intact poly(A)+ RNA from 4 M GuSCN-lysed cell extracts using a LNA-substituted oligo(dT) affinity probe, based on the increased affinity of LNA-T for complementary poly(A) tracts. In addition, we demonstrate that use of a LNA-substituted oligo(dT) probe enables efficient isolation of poly(A)+ RNA from extracted total RNA samples in a low salt binding buffer.
MATERIALS AND METHODS
Determination of the duplex melting temperatures
The melting temperatures of the LNA/RNA and the corresponding DNA/RNA duplexes were determined as described by Wahlestedt et al. (9) using 1.0 μM concentrations of the two complementary oligonucleotides in a medium salt buffer (10 mM sodium phosphate buffer, pH 7.0, 100 mM NaCl, 0.1 mM EDTA).
In vitro synthesis of polyadenylated yeast ACT1 and THI4 spike RNAs
Genomic DNA was prepared from Saccharomyces cerevisiae wild type (BY4741, MATa, his31, leu20, met150, ura30) (EUROSCARF) using the Nucleon MiY DNA extraction kit (Amersham Biosciences, UK) according to the manufacturer’s instructions. Amplification of the 3'-end of the ACT1 (YFL039C) and THI4 (YGR144W) open reading frames (ORFs) was performed by standard PCR using yeast genomic DNA as template. In the first PCR step, a forward primer containing a restriction enzyme site and a reverse primer containing a universal linker sequence were used and 20 bp was added to the 3'-end of the ORFs next to the stop codon. In the second step the reverse primer was exchanged with a nested primer containing a poly(dT)20 tail and a restriction site. The actin amplicon contained 720 bp of the ACT1 ORF plus a 20 bp universal linker sequence and a poly(dA)20 tail. The THI4 amplicon contained 723 bp of the THI4 ORF plus a 20 bp universal linker sequence and a poly(dA)20 tail. The PCR primers used were: YFL039C- Rev-Uni (5'-gatccccgggaattgccatgttagaaacacttgtggtgaacga-3') and YFL039C-For-EcoRI (5'-acgtgaattctttccatccaagccgttttg-3') for ACT1; YGR144W-Rev-Uni (5'-gatccccgggaattgccatgctaagcagcaaagtgtttcaaa-3') and YGR144W-For-EcoRI (5'-acgtgaattctcgccaagaacagaccagact-3') for THI4; Uni-polyT-BamHI (5'-acgtggatccttttttttttttttttttttgatccccgggaattgccatg-3') for both amplicons. The PCR products were digested with the restriction enzymes EcoRI and BamHI, ligated into the pTRIamp18 vector (Ambion, UK) using a Quick Ligation Kit (New England Biolabs, USA) according to the supplier’s instructions and transformed into Escherichia coli DH-5. DNA sequencing (ABI 377, Applied Biosystems, USA) was used to verify the plasmid constructs using M13 forward and reverse primers.
The yeast spike RNAs were synthesized using a MEGAscriptTM Kit (Ambion, UK) and BamHI-linearized ACT1 or THI4 plasmid clones as templates. The RNA preparations were treated with DNase I followed by purification using a RNeasy? Mini kit (Qiagen, Germany) according to the manufacturer’s instructions for in vitro RNA synthesis. The RNA quality was assessed by native agarose gel electrophoresis and the RNA concentration and purity were determined by measuring the absorbance at 260 and 280 nm.
Preparation of the magnetic particles for poly(A)+ RNA isolation
For each mRNA sample preparation, 60 μl of streptavidin-coated magnetic particles (Roche, Germany) were pipetted into an RNase-free siliconized tube (Ambion, UK). A magnetic separator (Roche, Germany) was used to remove the supernatant. An aliquot of 100 μl of 1 μg/ml yeast tRNA (Ambion, UK) diluted in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.5) was added and the mixture was incubated for 5 min at room temperature. The particles were washed in 100 μl of TE buffer and resuspended in 100 μl of 1x binding buffer . To the particles was added 200 pmol biotinylated DNA- or LNA-oligo(T) capture probe (Exiqon, Denmark) followed by incubation for 5 min at 37°C. The particles were washed twice in 1x binding buffer before collection and release to the RNA sample or lysed, cell-free sample preparation extract.
Tosyl-activated Dynabeads M-280 (Dynal, Norway) were washed in 0.1 M borate buffer, pH 9.5. To 500 μl of particles was added 10.5 nmol NH2-modified DNA- or LNA-oligo(T) capture probe , followed by incubation for 20 h at 37°C with slow end-over-end rotation. After incubation, the Dynabeads were collected with a Magnetic Particle Concentrator (MPC) (Dynal). The tube was placed in the MPC for 5 min to allow the beads to be collected by the magnet in it and the supernatant was discarded while the tube was in the MPC. The coated beads were then washed, using the MPC, twice for 10 min in 25 mM sodium citrate, 0.5% N-lauryl sarcosine and then overnight at 37°C with 500 μl of 220 mM Tris, pH 8.0, 0.05% N-lauryl sarcosine, 1 mM EDTA, pH 8.0. The coated beads were collected with the MPC, the supernatant discarded and the beads were resuspended in the GuSCN lysis buffer at a concentration of 30 mg/ml.
Yeast spike RNA recovery assays
The yeast spike RNA samples were denatured for 10 min at 65°C followed by quenching on ice for 10 min. For the GuSCN step gradient recovery assay 0.5 μg aliquots of the THI4 spike RNA were mixed in 100 μl of GuSCN lysis buffer (25 mM sodium citrate) (J.T. Baker, USA), pH 7.0, 0.5 g/100 ml sodium N-lauroyl sarcosinate) and 0, 0.5, 1, 2 or 4 M GuSCN (Sigma, USA), respectively. For capture in NaCl binding buffer, the ACT1 spike RNA samples were prepared by mixing 0.5 or 1 μg spike RNA in a final volume of 50 μl of DEPC-treated H2O (Ambion, UK) and 50 μl of 2x binding buffer , with the following NaCl concentrations in the 2x binding buffer; 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 M, respectively. All the samples were subsequently used in the poly(A)+ RNA isolation protocol with oligo(dT) and LNA-oligo(T) capture. The magnetic particles were transferred to the spike RNA mixture and incubated for 10 min at 37°C in an Eppendorf Thermomixer at 400 r.p.m. (Eppendorf, Germany). The particles were collected and released into the wash buffer , re-collected and the washing step was repeated twice. Finally, the poly(A)+ RNA was eluted in 10 μl of DEPC-treated H2O by heating the sample at 65°C for 10 min, quenching on ice and removing the particles. For subsequent analysis, 3 μl of the sample and a standard dilution series of either THI4 or ACT1 were applied on a 1% native agarose gel. The gel was subjected to electrophoresis for 20–30 min at 7 V/cm and then quantified on a Typhoon 9200 Imager (Amersham Biosciences, USA).
Yeast cultures and extraction of yeast total RNA
Saccharomyces cerevisiae wild type (BY4741, MATa, his31, leu20, met150, ura30) and a hsp78 mutant strain (BY4741, MATa, his31, leu20, met150, ura30, YDR258c::kanMX4) (EUROSCARF) were grown in YPD medium at 30°C until the A600 of the cultures reached 0.8. Half of the cultures were collected by centrifugation and resuspended in 1 vol of 40°C pre-heated YPD. Incubation was continued for an additional 30 min at 30 or 40°C for the standard and heat-shocked cultures, respectively. Cells were harvested by centrifugation and stored at –80°C. Yeast total RNA was extracted using a FastRNA Kit-RED (BIO 101, USA) according to the manufacturer’s instructions. The quantity and quality of the total RNA preparations were assessed by standard spectrophotometry or using a NanoDrop ND-1000 (NanoDrop, USA) combined with agarose gel electrophoresis.
Isolation of yeast poly(A)+ RNA
An aliquot of 100 μg yeast total RNA was combined in a final volume of 50 μl of 1x binding buffer and heated for 30 min at 65°C, 700 r.p.m. The tube was spun briefly and quenched on ice for 10 min. To the total RNA sample was added 200 pmol biotinylated DNA-oligo(dT)20 or LNA_2.T capture probe and the samples were incubated for 15 min at 37°C, followed by addition of streptavidin-coated particles and incubation of the sample preparation for 10 min at 37°C, 400 r.p.m. The magnetic particles were collected and released into the wash buffer, re-collected and the washing step was repeated twice. Finally, the poly(A)+ RNA was eluted in 10 μl of DEPC-treated H2O by heating the sample at 65°C for 10 min, quenching on ice and removing the particles.
Caenorhabditis elegans cultures
The C.elegans N2 Bristol strain was grown in S-medium, with E.coli NA22 food, at 23°C and was cleaned by standard sucrose density methods as described (10). Caenorhabditis elegans mixed stage worms were harvested and resuspended in 4 vol of GuSCN lysis buffer , immediately frozen in liquid nitrogen and stored at –80°C.
Isolation of C.elegans poly(A)+ RNA from 4 M GuSCN-lysed worm extracts
Caenorhabditis elegans samples stored in the GuSCN lysis buffer (see above) were thawed and the wet weight was calculated by removing the supernatant (centrifugation for 2 min at 4000 g) and weighing. The pellet was subsequently resuspended in the same volume. The C.elegans mixed stage worm aliquots were spun for 2 min at 4000 g and mixed with 200 μl of GuSCN lysis buffer. Quartz sand was added to the C.elegans samples and the samples were disrupted for 2 min on ice using a pestle. The extract was heated for 30 min at 65°C, 700 r.p.m., incubated on ice for 10 min and briefly spun down. The soluble, 4 M GuSCN-lysed C.elegans extract was transferred to a clean, RNase-free tube containing the LNA_2.T or DNA-oligo(dT)20 capture probe coupled to magnetic particles and incubated for 5 min at 37°C, 400 r.p.m. for poly(A)+ RNA selection. The particles were re-collected and released into the wash buffer and the washing step was repeated twice. Finally, the poly(A)+ RNA was eluted in 50 μl of DEPC-treated H2O. The samples were incubated for 10 min at 65°C and quenched on ice for 10 min to release the polyadenylated RNA from the particles. The particles were removed and the supernatant was spun briefly (1 min at 16 100 g) and transferred to a clean RNase-free siliconized tube. The isolated poly(A)+ RNA was ethanol precipitated by adding 0.1 vol 3 M NaOAc (Ambion, UK), 150 μg/ml Glycogen Carrier (Ambion, UK) and 2.5 vol 96% ethanol to the tube and stored at –20°C overnight. After spinning for 30 min at 16 100 g at 4°C the supernatant was removed and the pellet washed with ice-cold 70% ethanol and air dried. The pellet was dissolved in 10 μl of DEPC-treated H2O.
Isolation of poly(A)+ RNA from human K562 and vincristine-resistant K562/VCR cells
The K562 leukemia cells (a human erythroleukemia cell line derived from a chronic myeloid leukemia patient in blast crisis) were cultured in HEPES-buffered RPMI 1640 medium containing 5% newborn calf serum, 1% L-glutamine, 100 U penicillin and 100 mg/ml streptomycin (all from Gibco BRL Life Technologies, Denmark). All cultures were maintained at 37°C in an atmosphere of 95% air and 5% CO2. The resistant cells were kept in sub-lethal concentrations of drug. Wild-type K562 cells were used to develop the vincristine-resistant cell line. The cells were cultured at increasing drug concentrations for 35 passages. The drug resistance levels were measured by a clonogenic assay (11). In brief, single cell suspensions with the desired drug concentration were plated in soft agar containing sheep red blood cells and 2-mercaptoethanol. The plates were exposed continuously to the drugs and the experiments were performed in triplicate. The IC50 values were defined as the concentrations inhibiting 50% of colony formation, while the relative resistance was defined as the ratio between the IC50 value for the resistant line and the wild-type cell line. The K562/VCR cell line is 152 times more resistant to the chemotherapeutic drug vincristine compared to drug-sensitive K562. The cell cultures were harvested by centrifugation, the supernatant was removed and the pellets were frozen in N2 and stored at –80°C until use. The pellets were subsequently resuspended in the 4 M GuSCN lysis buffer containing 10 mM dithiothreitol (DTT) (107 cells/100 μl). Quartz sand was added and the samples were treated as described for the worm extracts. The human cell extracts were diluted to 106 cells/50 μl in the GuSCN lysis buffer containing 10 mM DTT before incubating with the LNA_2.T or DNA-oligo(dT)20 capture probe coupled to magnetic particles (5 min at 37°C) for poly(A)+ RNA selection. The particles were re-collected and released into wash buffer , the washing step being repeated twice. The last two washing steps were without sodium N-lauryl sarcosinate. Finally, the poly(A)+ RNA was eluted twice in 10 μl of DEPC-treated H2O by incubating for 10 min at 65°C and quenched on ice for 10 min to release the polyadenylated RNA from the particles. The eluates were pooled and treated as described above.
RT–PCR and quantitative real-time RT–PCR assays
An aliquot of 100 ng poly(A)+ RNA from mixed stage C.elegans worms was combined with 5 μg oligo(dT)12–18 primer (Amersham Biosciences, UK) and heated for 10 min at 70°C, followed by quenching on ice. First strand cDNA was synthesized by transferring the mixture to 20 μl of cDNA synthesis reaction containing 50 mM Tris–HCl (pH 8.3 at room temperature), 75 mM KCl, 3 mM MgCl2, 10 mM DTT (Invitrogen, USA), 1 mM each dATP, dCTP, dGTP and dTTP (Amersham Biosciences, USA) and 20 U Superasin (Ambion, UK) and incubating for 5 min at 37°C. An aliquot of 200 U SuperScriptTM II RT (Invitrogen, USA) was added and the reaction mixture was incubated for 30 min at 37°C and 30 min at 42°C. An additional 200 U SuperScriptTM II RT was added and the incubation at 42°C was prolonged for 1 h. Finally, the reaction was diluted five times in DEPC-treated H2O and heated for 5 min at 70°C and the oligo(dT)12–18 primer was removed on a Sephacryl S-400 HR spin column (Amersham Biosciences, UK) according to the manufacturer’s instructions. First strand cDNA was generated from poly(A)+ RNA isolated from human K562 and K562/VCR cells essentially as described above, except that 5 μg random hexamers were used instead of the oligo(dT)12–18 primer. The excess primer was removed on a Microcon YM-30 filter unit (Millipore, USA).
Four PCR fragments were amplified from first strand worm cDNA using gene-specific primer sets for: (i) C.elegans 26S rRNA (GenBank accession no. Z92784 ), 5'-gccagaggaaactctggtggaagtcc-3' and 5'-agcctcccttggtgttttaagggccg-3'; (ii) ?-actin (GenBank accession no. Z71181 ), 5'-ggattcgcaagggcgaaaggtgattg-3' and 5'-gctttattccaagtttggccatac-3'; (iii) let-2 (GenBank accession no. AAA96216 , 5'-gatcgaattcctccaggagagaagggagatg-3' and 5'-gatcaagcttatctctt cctgggtatccagctt-3'; (iv) T01D3.3 (GenBank accession no. Z81110 ), 5'-gatcgaattcatgatacctgcagatgtgcctgcctac-3' and 5'-gatcgagctccaatggatgttcacagtgttgctcg-3'. PCR reactions (25 μl) were carried out by mixing 15 mM Tris–HCl, pH 8.0, 50 mM KCl (GeneAmp Gold buffer; PE Biosystems, USA), 2.5 mM MgCl2, 200 μM each dATP, dCTP, dGTP and dTTP, 0.4 mmol/l forward primer; 0.4 mM reverse primer; 1.25 U (0.25 μl of a 5 U/μl solution) AmpliTaq Gold polymerase (PE Biosystems, USA) and 1 μl of cDNA as template. After an initial 5 min denaturation step at 95°C, 30 cycles of PCR were carried out (45 s at 95°C, 30 s at 60°C and 90 s at 72°C), followed by extension at 72°C for 10 min. The PCR products were analysed by native agarose gel electrophoresis. The 26S rRNA PCR fragment was purified by agarose gel electrophoresis followed by a Qiaquick PCR purification kit (Qiagen, Germany) for probe labelling according to the supplier’s instructions. Two PCR fragments were amplified from first strand human K562 cell line cDNA using gene-specific primer sets for: (i) human mdr1 (GenBank accession no. NM_000927 ), 5'-ttctagcccttggaattatttcttt-3' and 5'-ctgatattttggctttggcata-3'; (ii) ?-actin (GenBank accession no. NM_001101 ), 5'-tgccctgaggcactcttc-3' and 5'-cgatccacacggagtacttg-3'. The PCR reactions and cycling conditions were as described above.
Quantitative real-time PCR assays for the human mdr1 and ?-actin mRNAs were performed using 1x TaqMan Universal PCR Master Mix, No AmpErase UNG and 1x TaqMan Assays-on-Demand, Gene Expression Product for the mdr1 mRNAs (Assay ID Hs00184491_m1) or 1x TaqMan Pre-Developed Assay Reagent endogenous control for the ?-actin mRNA (TaqMan PDAR human ACTB, catalogue no. 4310881E) in an ABI PRISM 7000 Sequence Detection System as follows: 10 min denaturation at 95°C, followed by 50 cycles of denaturation for 15 s at 95°C and annealing and elongation for 1 min at 60°C using 2 μl of the diluted cDNA reaction from above as template in a final volume of 25 μl. The relative gene expression level of the mdr1 mRNA in the two human leukemia cell lines was calculated as described in User Bulletin no. 2 for the ABI PRISM 7700 Sequence Detection System. The expression data obtained represent average values from a minimum of three replicate experiments. All samples were normalized using the TaqMan PDAR human ACTB endogenous control for detection of the ?-actin mRNA level treated as an internal control. Unless otherwise stated, all reagents were purchased from Applied Biosystems (USA).
Northern blot analysis
Aliquots (500 ng/sample) of the LNA oligo_2.T-selected poly(A)+ RNA samples from wild type yeast cells and heat shocked wild type and hsp78 mutant cells, respectively, were subjected to electrophoresis in a 1.5% agarose–2.2 M formaldehyde gel (12) and blotted onto Hybond-N nylon membrane (Amersham Biosciences, UK) with 10x SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.0) as transfer buffer (13). A 756 bp PCR amplicon of the yeast HSP78 gene was 32P-labelled (>5 x 108 c.p.m./μg) by random priming (MegaprimeTM DNA labelling system; Amersham Biosciences, UK) and purified according to the manufacturer’s recommendations. Redivue dCTP (3000 Ci/mmol) was purchased from Amersham Biosciences (UK). The radioactively labelled probe was hybridized to the filter at 42°C for 18 h in ULTRA-hybTM (Ambion, UK). The filter was washed twice in 2x SSC, 0.1% SDS at 42°C for 5 min, then twice in 0.1x SSC, 0.1% SDS at 42°C for 15 min. After autoradiography on a Storage Phosphor screen (Amersham Biosciences, UK) and image analysis quantification using a Typhoon 9200 scanner (Amersham Biosciences, UK), the probe was removed from the filter according to the manufacturer’s instructions. The filter was rehybridized with a 748 bp PCR amplicon of the yeast ACT1 gene. 32P-labelling of the probe, hybridization to the filter and the washing steps were identical to those described for the HSP78 probe.
Aliquots of the LNA-oligo_2.T and DNA-oligo(dT)20 selected worm poly(A)+ RNA samples (4 μl/sample, 2.8, 5.5, 11, 22 and 44 mg worm extract, respectively) together with 10 μg C.elegans total RNA from worm embryos were subjected to northern blot analysis as described above. A 483 bp PCR amplicon of the RPL-21 cDNA (GenBank accession no. AAA27951 (a gift from M. Zagrobelny, University of Copenhagen, Denmark) was 32P-labelled (>5 x 108 c.p.m./μg). Hybridization to the filter and the washing steps were performed as described above. The filter was sequentially rehybridized with the let-2, mouse GAPDH (Ambion, USA) and 989 bp 26S rDNA fragments. 32P-labelling of the probes, hybridization to the filter and the washing steps were identical to those with the RPL-21 probe.
Poly(A)+ RNA aliquots of the LNA_2.T and DNA-oligo(dT)20 selected human K562 and K562/VCR samples were subjected to northern blot analysis as described above, except that the agarose gel was 1%. The mouse GAPDH (Ambion, USA) cDNA fragment was 32P-labelled and hybridized to the filter as described above.
RESULTS AND DISCUSSION
Total, cellular RNA is typically extracted from cells and tissues using strong chaotropic agents, which lyse the cells with simultaneous denaturation of endogenous RNases (2,3), followed by poly(A)+ RNA selection from the purified total RNA (1). The chaotropic salt GuSCN is widely used to lyse cells and release nucleic acids into solution due to its ability to effectively denature secondary/tertiary protein and nucleic acid structures (14). In addition to efficient cell lysis, its use in extraction buffers at a high concentration thus also leads to concomitant inhibition of endogenous proteases and nucleases, including RNases (2,15). In order to be effective both as a chaotrope and an RNase inhibitor, GuSCN has to be used at a high concentration of at least 4 M (16). In turn, this dramatically decreases the thermal stability of oligonucleotide duplexes, including dT–A duplexes exploited when using oligo(dT) affinity chromatography in poly(A) selection (17). Consequently, DNA-oligo(dT) chromatography has to date only been demonstrated at low concentrations of GuSCN, which is not sufficient for RNase inhibition (18,19).
To evaluate the hybridization properties of LNA-substituted oligo(dT) affinity probes, different LNA-oligo(T) oligonucleotides were designed and synthesized (Table 1). The duplex thermal stabilities of the different dT–A duplexes with complementary RNA were determined by measuring the melting temperatures (Tm). Substitution of a DNA-oligo(dT)20 oligonucleotide by LNA-T resulted in significantly increased thermal duplex stabilities in all LNA-oligo(T) designs measured, corresponding to an increase in melting temperature ranging from +2.8 to +6.0°C per LNA thymidine monomer. This is in good agreement with previous results reported for extensively modified oligothymidylate LNA oligonucleotides (20). Among the LNA-T probes, the fully substituted LNA-T20 and LNA-T15 oligonucleotides showed a Tm of >95°C, indicating that their exceptionally high thermal stability would not allow efficient elution of the captured poly(A)+ RNA from the affinity ligand. By comparison, LNA_2.T, in which every second thymidine was substituted with LNA-T, showed a Tm of 70.8°C and an increase of 30°C compared to the DNA control probe. Thus, the LNA_2.T affinity probe represented an adequate compromise between increased duplex thermal stability and melting of the dA–T duplexes in elution buffer. In our initial experiments the efficiency of all the different LNA-T-substituted affinity probes (Table 1) in poly(A)+ RNA isolation was assessed along with the reference DNA-oligo(dT)20 probe using polyadenylated, in vitro synthesized yeast spike RNAs. The recovery of the spike RNAs from GuSCN-containing binding buffer was consistently highest using the LNA_2.T affinity probe (data not shown). Thus, the LNA-oligo_2.T probe was chosen for all further experiments.
Table 1. Thermal duplex stabilities of different LNA-substituted oligo(T) affinity probes
The recovery of polyadenylated yeast THI4 spike RNA directly from a chaotropic lysis buffer was assessed using either the LNA_2.T or the reference DNA-oligo(dT)20 probe by increasing the final GuSCN concentration stepwise from 0 to 4 M GuSCN (Fig. 1A). The spike RNA recovery was low with the DNA control over the whole GuSCN concentration range (0.5–4 M) (Fig. 1A). The optimal GuSCN concentration for the reference DNA-oligo(dT)20 probe was found to be 0.5 M GuSCN (20% spike RNA recovery), in accordance with previous results reported with oligo(dT) chromatography (18). In contrast, the THI4 spike RNA recovery with the LNA_2.T probe was unaffected by increasing the GuSCN concentration in the binding buffer, showing comparable yields of 80% over the entire range from 0.5 to 4 M GuSCN. The results thus implied that the LNA_2.T affinity probe would be useful in direct isolation of poly(A)+ RNA from cell or tissue extracts lysed in 4 M GuSCN buffer, which effectively inhibits endogenous RNases.
Figure 1. The impact of (A) GuSCN and (B) NaCl concentration on the recovery of yeast spike RNAs. (A) Aliquots (500 ng) of the 0.8 kb yeast THI4 spike RNA were captured at increasing final GuSCN concentrations in binding buffer from 0 to 4 M GuSCN using the reference DNA-oligo(dT)20 and LNA_2.T affinity probes, electrophoresed in a 1% agarose gel stained with Gelstar. Lanes 1–6, THI4 spike RNA standard curve (STD) with 250, 125, 62.5, 31.3, 15.6 and 7.8 ng/lane, respectively; lanes 7–11, THI4 spike RNA captured by the reference DNA-oligo(dT)20 in 0, 0.5, 1, 2 and 4 M GuSCN, respectively; lanes 12–16, THI4 spike RNA captured by the LNA_2.T affinity probe in 0, 0.5, 1, 2 and 4 M GuSCN, respectively. A 1 kb DNA ladder (Invitrogen, USA) was used as a size marker. Recovery (%) of the yeast THI4 RNA by the reference DNA-oligo(dT)20 (open bars) and the LNA_2.T (solid bars) affinity probes, respectively, at increasing GuSCN concentrations was calculated by image analysis of the agarose gel. (B) Aliquots (1 μg) of the 0.8 kb yeast ACT1 spike RNA were captured at increasing final salt concentrations in binding buffer from 0 to 0.5 M NaCl using the reference DNA-oligo(dT)20 and LNA_2.T affinity probes and electrophoresed in a 1% agarose gel stained with Gelstar (not shown). Recovery (%) of the yeast ACT1 RNA by the reference DNA-oligo(dT)20 (open bars) and the LNA_2.T (solid bars) affinity probes, respectively, at different NaCl concentrations (0–0.5 M) was calculated by image analysis of the agarose gel.
Next, we tested the same oligo(T) affinity probes in a spike RNA recovery assay employing different hybridization conditions in a non-chaotropic poly(A) binding buffer by varying the salt concentration range from 0 to 0.5 M NaCl (Fig. 1B). A high spike RNA recovery of 70–100% was observed for both the reference DNA-oligo(dT)20 and LNA_2.T affinity probes in the high salt concentration range 0.2–0.5 M NaCl in the binding buffer (Fig. 1B). However, in the low salt range 50–100 mM a significantly decreased recovery of the ACT1 spike RNA was observed with the reference DNA-oligo(dT)20 probe, while the recovery was still between 80 and 90% with the LNA-oligo(T) affinity ligand, indicating that a low salt, high hybridization stringency window could be employed in combination with the LNA-oligo(T) affinity probe without compromising the mRNA yield.
The validity of this conclusion was assessed by poly(A)+ RNA selection experiments with yeast RNA samples. Total RNA was extracted by standard methods from S.cerevisiae wild type, heat-shocked wild type and heat-shocked hsp78 mutant cells, respectively. The hsp78 mutant is a strain in which the HSP78 gene is deleted and thus HSP78 mRNA is not present in these cells (21). Yeast poly(A)+ RNA was subsequently isolated from the total RNA samples using the LNA_2.T affinity probe under low salt (100 mM) binding conditions and the quality was assessed by northern blot analysis (Fig. 2). The mRNA yield in the LNA_2.T-selected poly(A)+ RNA samples corresponded to a recovery of 2.6–3.8% of the input yeast total RNA. The hybridized northern blot revealed a prominent 2.3 kb message in the heat-shocked wild-type strain and a weakly hybridizing HSP78 mRNA in the wild-type control (Fig. 2). Furthermore, the HSP78 mRNA was 14-fold up-regulated as a response to the heat shock treatment in the wild type, whereas no HSP78 transcript was detected from the heat-shocked hsp78 mutant strain mRNA sample, as expected. By comparison, the ACT1, which was used as a loading control in the northern analysis, revealed a 1.1 kb mRNA of comparable intensity in all three yeast samples (Fig. 2). All the isolated yeast mRNA samples were fully intact as judged by the northern blot analysis. In conclusion, the LNA_2.T affinity probe is highly efficient in isolation of poly(A)+ RNA at a low salt concentration of 100 mM NaCl in the poly(A) binding buffer. We therefore anticipate that low salt, high stringency hybridization conditions would be highly preferable compared to the standard method employing 0.5 M NaCl in the binding buffer, due to the enhanced destabilization of secondary structures in the mRNA.
Figure 2. Northern blot analysis of yeast poly(A)+ RNA isolated by the LNA_2.T affinity probe under low salt binding conditions. Yeast poly(A)+ RNA was isolated from wild type, wild type heat shocked and hsp78 mutant heat shocked total RNA and probed sequentially with 32P-labelled fragments for the yeast HSP78 and ACT1 genes, respectively.
To assess whether the LNA-oligo(T) capture method would allow mRNA sample preparation directly from cell or tissue extracts lysed in 4 M GuSCN buffer, we isolated poly(A)+ RNAs from C.elegans worm extracts using the LNA_2.T affinity probe in comparison with the reference DNA-oligo(dT)20 probe. The isolated worm poly(A)+ RNA samples were subsequently evaluated by northern blot analysis (Fig. 3). The cDNA probes for the C.elegans let-2, ?-actin and RPL-21 mRNAs readily detected single mRNA species in the worm samples selected with the LNA_2.T affinity probe, whereas the samples captured with the reference DNA-oligo(dT)20 probe showed only weakly hybridizing fragments on the northern blot (Fig. 3). Moreover, the message levels for all three transcripts increased as a function of increased worm extract sample size in the LNA_2.T selected samples, indicating that poly(A) selection by the LNA-substituted affinity probe is specific for polyadenylated mRNAs. This is in good agreement with the results obtained with the yeast spike RNA recovery assay (Fig. 1A) and implies that the reference DNA-oligo(dT)20 probe is unable to form stable dT–A duplexes in the presence of 4 M GuSCN. None of the isolated worm mRNA samples showed degradation by RNases, as judged by the northern analysis. Re-probing the same northern filter with a 32P-labelled DNA fragment for the C.elegans 26S rRNA probe revealed a prevalent, strongly hybridizing 3.5 kb rRNA species in the control total RNA sample, corresponding to the 26S rRNA, while only weakly hybridizing 26S rRNA fragments were detected in the poly(A) selected worm mRNA samples (Fig. 3). Estimation of the mRNA levels based on the data obtained by northern blot image analysis for the three C.elegans transcripts (Fig. 3) implies that the mRNA yield by the LNA_2.T affinity probe is more than 50-fold higher compared to the DNA control probe, when using the same worm extract sample size. On the other hand, the ratios of the let-2, GAPDH and RPL-21 mRNAs to the 26S rRNA were estimated to be 1.3, 35 and 191, respectively, for the LNA-oligo(T)-selected mRNA samples compared to those of the DNA-oligo(dT)20 control samples, where the ratios were estimated to be 0.1, 7 and 17 thus further supporting the notion that LNA_2.T is highly specific for polyadenylated mRNAs.
Figure 3. Direct isolation of poly(A)+ RNA from 4 M GuSCN-lysed C.elegans worm extracts using LNA-oligo(T) capture. Northern blot analysis of the poly(A)+ RNA samples from 4 M GuSCN-lysed mixed stage C.elegans worm extracts is shown as a function of increasing sample size (2.8, 5.5, 11, 22 and 44 mg wet weight), selected using the DNA-oligo(dT)20 or the LNA_2.T affinity probe. The same filter was sequentially hybridized with 32P-labelled DNA fragments for the C.elegans RPL-21, let-2 and GAPDH mRNAs and cytosolic 26S rRNA. Ten micrograms of total RNA isolated from C.elegans embryos was used as a control.
Next, we subjected the isolated worm poly(A)+ RNA samples to reverse transcription (RT) PCR assays for C.elegans mRNAs representing three abundance classes, in accord with previously published, compiled data from genome-wide expression profiling in C.elegans, reporting the following expression levels from embryos to adult worms for the aforementioned three messages; 35–207 p.p.m. for let-2, 6–21 p.p.m. for ?-actin and 3–8 p.p.m. for T01D3.3 (22). The LNA_2.T- and reference DNA-oligo(dT)20-captured poly(A)+ RNA samples were reversed transcribed to first strand cDNA using standard methods followed by PCR amplification of cDNA fragments for C.elegans ?-actin, let-2 and T01D3.3 (23), respectively. Analysis of the PCR products obtained by RT–PCR of LNA_2.T-selected mRNA revealed single, prominent PCR amplicons of the expected size with each of the three primer pairs (data not shown). By contrast, only a weak PCR amplicon for the let-2 cDNA was detected by RT–PCR of the DNA-oligo(dT)20-selected control worm mRNA, in accordance with the low yield at 4 M GuSCN concentration.
In order to apply the LNA-oligo(T) capture method to mRNA sample preparation from human cells, we isolated poly(A)+ RNAs directly from 4 M GuSCN-lysed K562 and K562/VCR leukemia cells, respectively, using the biotinylated LNA_2.T probe along with a 5' NH2-modified LNA_2.T probe, which allows covalent coupling of the affinity probe onto pre-activated magnetic particles. The yield was 300 ng poly(A)+ RNA from 106 K562 cells with both LNA probes, whereas no mRNA could be captured with the DNA-oligo(dT)20 control probes, in accord with our previous results. Northern blot analysis of the poly(A)+ RNA samples revealed a single 1.3 kb mRNA species for the human GAPDH gene in the K562 and K562/VCR sample preparations selected with both LNA_2.T affinity probes (Fig. 4A).
Figure 4. Analysis of poly(A)+ RNA isolated directly from 4 M GuSCN-lysed human K562 and K562/VCR erythroleukemia cells by LNA-oligo(T) capture. (A) Northern blot analysis of the poly(A)+ RNA samples selected from 4 M GuSCN-lysed human K562 (1) and K562/VCR (2) cells, respectively, using the 5'-biotinylated or 5'-NH2-modified LNA_2.T affinity probe and the corresponding DNA-oligo(dT)20 control probes. The filter was hybridized with a 32P-labelled DNA fragment for mouse GAPDH mRNA. (B) Approximately 100 ng poly(A)+ RNA purified from the human K562 (1) and K562/VCR (2) cell lines was used as template for RT–PCR assays for human mdr1 and ?-actin. The amplicon sizes were 256 and 738 bp for the ?-actin and mdr1 mRNAs, respectively. The RT–PCR products were electrophoresed in a 1% native agarose gel and visualized by staining with Gelstar. A negative PCR control without template was performed for each assay. (C) Representative amplification plots of quantitative real-time RT–PCR assays for the human mdr1 transcript using mRNA samples isolated from human erythroleukemia cells as template. The poly(A)+ RNAs were selected either using the biotinylated LNA_2.T affinity probe from K562 cells (open triangle) and K562/VCR cells (solid triangle) or the 5'-NH2-modifed LNA_2.T affinity probe from K562 cells (open square) and K562/VCR cells (solid square). The plots relate the PCR cycle number to the change in detected, baseline-corrected fluorescence (Rn). The small, solid circles depict the fluorescence generated from the no template control reaction.
Subsequent RT–PCR assays for the human ?-actin mRNA revealed single cDNA fragments of the expected size in all four LNA_2.T-selected mRNA templates, whereas no PCR products were detected after 30 cycles of amplification from the DNA-oligo(dT)20-selected control samples (Fig. 4B). In contrast, RT–PCR for the human multidrug resistance gene mdr1 generated the 738 bp PCR amplicon in the K562/VCR cell line, but not in the drug-sensitive K562 cell line, implying that the mdr1 gene is overexpressed in K562/VCR cells, presumably reflecting their significantly increased resistance to the chemotherapeutic drug vincristine. We therefore decided to assess relative expression of the human mdr1 gene in the two K562 cell lines by quantitative real-time RT–PCR using a pre-validated TaqMan assay and ?-actin as an internal control. First, we used aliquots of the isolated poly(A)+ RNA samples as templates for reverse transcription, followed by a 5-fold dilution of the cDNA synthesis reaction prior to quantitative real-time PCR. Subsequent real-time PCR assays revealed an average increase of four orders of magnitude in mdr1 expression relative to ?-actin mRNA in the vincristine-resistant K562/VCR cell line compared to the sensitive K562 cells (Table 2 and Fig. 4C), which is in good agreement with the standard RT–PCR results. By comparison, Fujimaki et al. (24) have reported relative mdr1 expression levels of 0.21–6.7 in different K562/VCR cell lines, whereas the level was <0.0041 in other drug-sensitive cell lines, as assayed by real-time RT–PCR using GAPDH as an internal control. Our expression data were highly comparable with poly(A)+ RNA obtained with both LNA_2.T affinity capture methods (Fig. 4C). Given the average yield of 300 ng per 106 cells and the 5-fold dilution of the cDNA synthesis reaction, a single LNA-oligo(T) sample preparation would allow quantification of 33 different mRNAs in triplicate using real-time PCR assays. The fact that we were successful in substituting the biotinylated LNA_2.T affinity probe by the NH2-modified LNA_2.T probe strongly suggests that the LNA-oligo(T) method is amenable to automation for streamlined, high throughput expression profiling by real-time PCR by covalently coupling the probe to solid, pre-activated surfaces, such as microtitre plate wells or magnetic particles.
Table 2. Relative expression of the multidrug resistance gene mdr1 in human vincristine-resistant K652/VCR cells compared to drug-sensitive K562 cells
The results presented here confirm earlier reports (18) that optimal hybridization conditions for DNA-oligo(dT)20 in a GuSCN-containing lysis buffer is between 0.5 and 1 M GuSCN, whereas capture is highly inefficient at higher GuSCN concentrations. Hence, several poly(A)+ RNA isolation procedures initiate with crude cell lysates homogenized in the presence of 4 M GuSCN (25), followed by dilution of the GuSCN lysis buffer prior to poly(A)+ RNA selection by oligo(dT) capture. Thus, the existing DNA-oligo(dT) and PNA-T (5) methods have the disadvantage of selecting the poly(A)+ RNA under moderate chaotropic salt concentration where RNase inhibition could be inadequate. Furthermore, handling of many sample preparations simultaneously, e.g. in high throughput expression analyses, becomes more labour intensive and time consuming due to the additional dilution step. In contrast, the results presented in this study demonstrate that the LNA-substituted LNA_2.T affinity probe is capable of selecting poly(A)+ RNA in the presence of 4 M GuSCN in the cell lysis buffer due to its exceptionally high affinity for complementary poly(A) tracts. This enables direct and fast poly(A)+ RNA capture under conditions allowing complete inhibition of endogenous RNase activity, resulting in highly intact mRNA samples. We were successful in exploiting the biotin–streptavidin coupling chemistry in our mRNA isolation procedure by limiting the hybridization time to 5 min, in order to prevent streptavidin denaturation even in the presence of 4 M GuSCN. This is in accord with previous studies reporting that streptavidin is highly resistant to denaturation by guanidine hydrochloride (26–28). Furthermore, we demonstrated the utility of the LNA-oligo(T) sample preparation method employing a 5' NH2-modified LNA_2.T affinity probe coupled covalently to pre-activated magnetic particles, thus overcoming the potential problem of denaturation by GuSCN.
In summary, we have demonstrated that the use of the LNA affinity probe LNA_2.T, in which every second thymidine residue is substituted with a LNA-T, enables efficient isolation of poly(A)+ RNA from 4 M GuSCN-lysed cell extracts as well as from total RNA in a low salt binding buffer, due to the exceptionally high affinity of LNA_2.T for complementary poly(A) tracts. On the other hand, the similar electrostatic properties of LNA compared to DNA and RNA oligonucleotides contribute to good aqueous solubility, facilitating use of the LNA_2.T affinity probe in mRNA sample preparation as part of an automated set-up. Thus, the results described here strongly suggest that the LNA-oligo(T)-based poly(A) selection method represents an attractive alternative to current mRNA sample preparation protocols, resulting in the recovery of highly enriched and intact poly(A)+ RNA, suitable for a wide range of downstream applications, including high throughput gene expression profiling by real-time quantitative PCR.
ACKNOWLEDGEMENTS
We thank Marianne S?s Ludvigsen, Marianne Bonde Mogensen and Mette Bj?rn at Exiqon for expert technical assistance. Also Pia Friis at the Department of Evolutionary Biology, University of Copenhagen and Marianne Fregil at the Laboratory of Oncology 54O5, Herlev University Hospital are thanked for their skilled technical support.
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