Which is a better starting sample for preparing RT-qPCR calibration curves, RNA or cDNA?
Which is a better starting sample for preparing RT-qPCR calibration curves, RNA or cDNA?
Calibration curves for RT-qPCR may be prepared by either of the following methods:
*Serial dilution of RNA, followed by reverse transcription and real-time PCR
*Serial dilution of cDNA (obtained by reverse transcription reaction), followed by real-time PCR
Since the two methods evaluate different parameters, it is important to choose a method appropriate for the experimental system being used. Calibration curves prepared from diluted RNA samples reflect differences in not only PCR amplification efficiency, but also differences in reverse transcription efficiency, which is dependent on the amount of RNA. PCR amplification efficiencies determined from such calibration curves may potentially differ from the actual efficiency.
For Absolute Quantification, the reverse transcription efficiency is critical. Therefore, use serially diluted RNA to prepare calibration curves (cDNA dilution is unsuitable).
For Relative Quantification, differences in reverse transcription efficiency can be corrected by assaying a reference gene, such as a housekeeping gene. The use of serially diluted cDNA is recommended for preparing calibration curves.