Sunday, May 16, 2010

Factors Contributing to the Deviation of RT-qPCR Efficiencies

By Ann L. Bohannon-Stewart

Research Problem
By Dr. X. Wang

RT-PCR (reverse transcription-polymerase chain reaction) is a method for the detection of mRNA expression levels. RT-PCR involves two steps: the RT reaction and PCR amplification. RNA is first reverse transcribed into cDNA using a reverse transcriptase. The resulting cDNA is used as templates for subsequent PCR amplification using primers specific for one or more genes. RT-PCR can also be carried out as one-step RT-PCR in which all reaction components are mixed in one tube prior to starting the reactions. One-step RT-PCR offers simplicity and convenience and minimizes the possibility for contamination.
Real time RT-PCR (RT-qPCR) is widely used in determination RNA expression in various tissues of various organisms. If properly validate, this convenient method can quantify specific RNA sequence in few hours, comparing with days needed by Northern blot analysis. RT-qPCR analysis is commonly performed in two steps or one step. In many reports, one step RT-qPCR using SYBR green chemistry is the method of choice.
Many gene expression studies using RT-qPCR method convert gene expression level with 2-ΔΔCt. While this conversion is convenient and appropriate when amplification efficiency is close 100%, problems arise when efficiency significantly deviates from 100%. We have experienced during primer validation analysis that RT-qPCR efficiency may appear to be much greater than 100%, sometime reproducibly as high as 470% using SYBR green one step RT-qPCR kit. In contrast, qPCR analysis using SYBR Greener kit (Invitrogen) showed efficiencies much closer to 100%. Large deviations severely limit quantification accuracy. It is necessary to determine the cause of the appeared high efficiency and to develop approaches to overcome the problem.
The unexpected high efficiency may be caused by several reasons. The first reason could be the chemistry of the kits that were used in the experiments. There are two different kits that were used. One is SYBR greener from Invitogen and the other is Quantect RT-PCR kit (Qiagen) kit. The protocol procedure and chemistry of the two different PCR kits could have been contributing factors.
The next theory is during the process of the running of the first 2 cycles of the RT PCR. The reverse transcriptase may not be completing the conversion of all the RNA to DNA. It could be possible that not all the RNA is getting converted into cDNA during the first step of the PCR process.
To determine the major cause of efficiency deviation, we are testing both kits and protocols from SYBR greener and Qiagen using RNA and DNA as starting materials. First, we will test whether PCR stage response linearly to the amount of initial DNA with SYBR green one step RT-PCR chemistry. Next, we will compare results of one step RT-PCR with two step PCR method. Third, we will optimize the time used in reverse transcription stege.