RT-PCR without RNA isolation.
Reverse transcription-PCR (RT-PCR) has traditionally required time-consuming RNA extraction and purification. This report demonstrates that one can completely avoid the RNA extraction step in RT-PCR by basing the comparison of samples on cell number rather than micrograms of total RNA. A new method for lysing cells while preserving RNA is described. RT-PCR is carried out (i) by rapidly freezing cells in the presence of ribonuclease inhibitor (RNase inhibitor) plus dithiothreitol and (ii) by using extracts of 250 or fewer cells directly in the RT-PCR assay. Aldolase mRNA, extracted by freeze-thawing cells in the presence of RNase inhibitor, was found to be stable at 42 degrees C for over three hours. Since the RT step can be completed within 1 h, there is minimal degradation of mRNA. This simple procedure avoids the use of harsh reagents, which may inhibit enzymes involved in RT-PCR, and produces results virtually identical to methods that employ guanidinium thiocyanate and phenol for RNA extraction. Optimized conditions for each parameter of the procedure are described that permit amplification of mRNA from as few as four cells.
Klebe
R J
RJ
University of Texas Health Science Center, San Antonio, USA.
Grant
G M
GM
Grant
A M
AM
Garcia
M A
MA
Giambernardi
T A
TA
Taylor
G P
GP
D005615:Freezing; D006801:Humans; D012516:Osteosarcoma / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000302:isolation & purification*; D012260:Ribonucleases / Q000037:antagonists & inhibitors; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D012260:Ribonucleases