Protocol

Semiquantitative Analysis of Arabidopsis RNA by Reverse Transcription Followed by PCR Using Mimics

Adapted from Arabidopsis: A Laboratory Manual (eds. Weigel and Glazebrook). CSHL Press, Cold Spring Harbor, NY, USA, 2002.

INTRODUCTION

This article describes the analysis and quantification of Arabidopsis RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). RNA is first reverse-transcribed into cDNA, which is then amplified by competitive PCR using a mimic. Multiple PCRs are performed using a constant amount of unknown cDNA and varying amounts of a mimic; that is, a template of known concentration that is close in size and GC content to the quantified target and that contains the same annealing sites for primers. Mimics are usually designed to produce PCR products ~10%-20% smaller or larger than the endogenous target. A mimic can be used as a DNA standard (as in this protocol), to be included after the reverse transcription reaction, or, even better, as an RNA standard. In the latter case, an RNA molecule must be generated from a vector designed for in vitro transcription, such as pGEM or pBluescript series vectors. The procedure described here is based on commercial kit protocols.