Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture.
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.
Jacobsen
Nana
N
Department of Functional Genomics, Exiqon, Bygstubben 9, DK-2950 Vedbaek, Denmark. jacobsen@exiqon.com
Nielsen
Peter Stein
PS
Jeffares
Daniel Charlton
DC
Eriksen
Jens
J
Ohlsson
Helle
H
Arctander
Peter
P
Kauppinen
Sakari
S
D000199:Actins / Q000235:genetics; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics; D002457:Cell Extracts / Q000235:genetics*; D002846:Chromatography, Affinity / Q000379:methods*; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D005800:Genes, Fungal / Q000235:genetics; D019450:Genes, MDR / Q000235:genetics; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D020014:K562 Cells; D008024:Ligands; D008280:Magnetics; D008863:Microspheres; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011061:Poly A / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000145:classification / Q000235:genetics; D012965:Sodium Chloride / Q000494:pharmacology; D013861:Thiocyanates / Q000494:pharmacology; D013936:Thymidine / Q000031:analogs & derivatives / Q000378:metabolism; D014750:Vincristine / Q000494:pharmacology
D000199:Actins; D002457:Cell Extracts; D015342:DNA Probes; D006146:Guanidines; D008024:Ligands; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013861:Thiocyanates; C477371:locked nucleic acid; D011061:Poly A; D012965:Sodium Chloride; C054436:guanidine thiocyanate; D014750:Vincristine; D013936:Thymidine