Efficient poly(A)+ RNA selection using LNA oligo(T) capture.
This chapter describes a method for the isolation of intact polyadenylated mRNA using LNA oligo(T) capture. The method enables efficient isolation of poly(A)(+) RNA directly from guanidinium thiocyanate (GuSCN)-containing cell or tissue extract by combining the design of biotinylated LNA oligo(T) capture probes with subsequent immobilization of the captured poly(A)(+) RNA onto streptavidin-coated magnetic particles. In contrast to DNA oligo-dT and polyT PNA based mRNA isolation techniques, the LNA oligo(T) capture method allows poly(A) selection in the presence of 4 M GuSCN cell lysis buffer, which is needed for efficient inactivation of endogenous RNases. In addition, LNA oligo(T) facilitates highly efficient poly(A)(+) isolation at elevated temperatures compared to standard oligo(dT) technology. The successful use of the LNA oligo(T) capture method in recovery of mRNA from human cells and the subsequent use of the mRNA in northern blotting analysis, RT-PCR and qRT-PCR are demonstrated.
Exiqon, Vedbaek, Denmark.
Nana
N
Jacobsen
Jens
J
Eriksen
Peter Stein
PS
Nielsen
D000818:Animals; D015152:Blotting, Northern; D017173:Caenorhabditis elegans; D005821:Genetic Techniques; D006801:Humans; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry*; D026723:Polyadenylation; D012333:RNA, Messenger / Q000737:chemistry / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D012333:RNA, Messenger; C477371:locked nucleic acid; C027903:oligo (dT)