RT-PCR WITHOUT RNA ISOLATION

Reverse transcription-PCR (RT-PCR) has traditionally required time-con suming RNA extraction and purification. This report demonstrates that one can completely avoid the RNA extraction step in RT-PCR by basing t he comparison of samples on cell number rather than micrograms of tota l 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 (i i) by using extracts of 250 or fewer cells directly in the RT-PCR assa y. Aldolase mRNA, extracted by freeze-thawing cells in the presence of RNase inhibitor, was found to be stable at 42 degrees C for over thre e hours. Since the RT step can be completed within 1 h, there is minim al degradation of mRNA. This simple procedure avoids the use of harsh reagents, which may inhibit enzymes involved in RT-PCR, and produces r esults virtually identical to methods that employ guanidinium thiocyan ate and phenol for RNA extraction. Optimized conditions for each param eter of the procedure are described that permit amplification of mRNA from as few as four cells.