Reverse-transcription quantitative PCR directly from cells without RNA extraction and without isothermal reverse-transcription: a 'zero-step' RT-qPCR protocol.

ABSTRACT: We describe an ultra-rapid and sensitive method to quantify gene expression levels in cultured cells. The procedure is based on reverse-transcription quantitative PCR (RT-qPCR) directly from cells, without RNA extraction and without an isothermal reverse-transcription step. Human neurons (Lund human mesencephalic cells) were lysed at different stages of differentiation, and the lysates were used directly as template for the combined RT-qPCR reaction. We detected a down-regulation of a proliferation marker and an up-regulation of neuronal dopaminergic genes expression. We were able to detect the reference gene target from as few as a single cell, demonstrating the application of the method for efficient amplification from small cell numbers. The data were fully in line with those obtained by the standard two-step RT-qPCR from the extracted total RNA. Our 'zero-step' RT-qPCR method proved to be simple and reliable with a total time from cell lysis to the end of the qPCR as short as 1.5?h. It is therefore particularly suitable for RT-qPCRs where large numbers of samples must be handled, or where data are required within short time.

SUBMITTER: Chovancova P 

PROVIDER: S-EPMC6977950 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature