Santosh P., Jul 2005
The RT-PCR method can be used not only to detect specific mRNAs but also to semi-quantitate their levels. Thus, one can compare levels of transcripts in different samples. This can be done in two different ways. One is to quantitate against levels of transcripts from a control, house-keeping gene (such as actin and GAPDH). (Transcription of house-keeping genes is believed to be unaffected by almost all experimental conditions.) The second method is to add an exogenous, primer-specific PCR template during PCR.
Quantitating against house-keeping gene transcript
The method involves reverse transcription using an oligo-dT or random hexamer primer. The resulting cDNA thus represents both house-keeping gene transcripts as well as specific transcripts one is quantitating. The RT reaction is then amplified in a pair of PCR series - one series is to amplify the house-keeping gene cDNA (using GAPDH-, etc., specific primers), and the other is for the specific cDNA of interest (in separate PCR, using gene-specific primers).
The different PCR tubes within each series are set up such that they either vary in the amount of template (amount of RT reaction product [cDNA]) or undergo PCR for different number of amplification cycles. This is because PCR amplification, though theoretically logarithmic, is not so at low or high number of amplification cycles. The logarithmic or exponential amplification usually occurs only during the middle cycles, and this depends on the concentration of target template. Comparison can therefore be done only during this phase.
After PCR, same volume of reaction products are electrophoresed on an agarose gel (preferably on the same gel). Images of stained DNA (PCR products) are then obtained and analysed by visual comparison or with computer software (like Image J). An example is shown below where semi-quantitation by visual inspection was done for expression of Fut9 gene in two cell lines, LEC12 and LEC29.
It can be seen that 1) for both cell lines, exponential amplification for both GAPDH and Fut9 occurs during cycles 33-42. 2) the GAPDH band intensities during those cycles suggest almost similar levels of template (cDNA) was used for noth cell lines. 3) the Fut9 levels are about 6-9 cycles 'behind' in LEC12 than LEC29.
1. One should do pilot experiments first to make sure that only single, specific products are obtained with the PCR primers and to get an idea of the range of cycles or RT reaction dilutions to use.
2. One should start with equal amounts of RNA for the RT reaction.
3. All reactions should be carried out at the same time, using reagents that are prepared as a supermix to minimize any variation.
4. PCR products should be run on the same gel, again to minimize any variations.
5. When quantitating relatively rare transcripts, it may be better to use for controls those house keeping genes whose transcripts are present at a low level. E.g., transcripts of aldolase A are expressed at a low level compared to those of GAPDH gene.
6. When doing PCR for different number of cycles, set up PCR reactions together, removing them one after one during the middle of the extension phase during the desired PCR cycle numbers. Then place them in a water bath at 65-75 deg C for a few minutes to ensure that extension is complete.
7. When doing PCR with different RT reaction volumes (usually a series of 2 or 3 fold dilutions), dilute the RT reaction using an RT reaction that had no RNA template so the different PCR reactions have the same volume of RT reaction (but different amounts of cDNA). This is necessary because components other than cDNA in the RT reaction (such as RT, DTT, etc.) can affect PCR efficiency.
Semi-quantitation using exogenous standards
One can have exogenous standards (either RNA or DNA) that can be amplified using the same primers that amplify the transcript of interest. The standards are designed to have the same sequence as that of the transcript of interest except that they are 40-50 nucleotides shorter or longer. Different amounts of the standard is used in different PCR reactions (if the standard is an RNA, it is added prior to the RT step) and amplified for same number of cycles. The exogenous standard competes with the endogenous template, thus this strategy is also refered to as competitive RTPCR.
Products are electophoresed and quantitated by software and an 'amount of standard' vs 'band intensity' plot is drawn.
In the example shown above, product 1 is from the standard and 2 is from the endogenous transcript. It can be seen that the amount of standard added in the PCR reaction for lane 5 roughly equals the transcript amount.
Also see Reverse transcription