Freezing of Isolated Cells Provides Free mRNA for RT-PCR Amplification

摘要

The most common methods for isolating intact RNA are modifications of the guanidinium thiocyanate method(1) or of the acid guanidinium thiocyanate-phenol-chloroform (AGPC) method (2). For certain applications, both methods are too time-consuming. The use of diethyl pyrocarbonate (DEPC) has been described as a fast method for obtaining RNA (4). Residual DEPC, however, is a likely candidate for inhibiting reverse transcription. In addition, the boiling of the cell lysate, performed to achieve degradation of DEPC into CO_2 and H_2O, will also destroy a portion of the cellular RNA. Therefore, we developed a very fast, chemical-free and sensitive method for making RNA available for reverse transcription polymerase chain reaction (RT-PCR) with specific primers.