Direct reverse transcription-PCR on oligo(dT)-immobilized polypropylene microplates after capturing total mRNA from crude cell lysates.
To simplify gene expression analysis, oligo(dT)-immobilized polypropylene microplates were used serially to capture mRNA, synthesize cDNA, and amplify specific genes. The amounts of immobilized oligonucleotide, hybridized mRNA, and synthesized cDNA were quantified fluorometrically using either Yoyo-1 or AttoPhos. The immobilized oligonucleotides captured approximately 40-55% of mRNA directly from crude cell lysates. Hybridized mRNA was then amplified by one-step reverse transcription (RT)-PCR with rTth polymerase or two-step PCR with initial cDNA synthesis followed by PCR, where the latter exhibited more sensitivity. In two-step RT-PCR, microplates can be reused for multiple PCRs with the same or different primer sets because synthesized cDNA was covalently attached to the plates at its 5' end. We believe this microplate may be acceptable as a platform for various mRNA expression analyses, including basic research, drug screening, and molecular toxicology, as well as for molecular pathological diagnostics.
Hamaguchi
Y
Y
Department of Pathology, University of California, Irvine 92612, USA.
Aso
Y
Y
Shimada
H
H
Mitsuhashi
M
M
D000818:Animals; D004247:DNA / Q000032:analysis; D018076:DNA, Complementary / Q000032:analysis / Q000138:chemical synthesis; D005914:Globins / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D008904:Miniaturization; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D011126:Polypropylenes; D012333:RNA, Messenger / Q000032:analysis*; D012334:RNA, Neoplasm / Q000032:analysis; D012335:RNA, Ribosomal / Q000032:analysis; D012343:RNA, Transfer / Q000032:analysis; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D011126:Polypropylenes; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012335:RNA, Ribosomal; D005914:Globins; D004247:DNA; D012343:RNA, Transfer