When carrying out cDNA synthesis using the SensiFAST™ cDNA Synthesis Kit, I prepared a NO-RT control by including water instead of reverse transcriptase (along with RNA up to 1μg and 5X TransAmp Buffer).
However, when this sample is included in my qPCR, amplification is seen when using some primers but not detected when using other separate primers. The ct values obtained from these NO-RT controls is similar to my samples.
Could it be my primers forming primer dimers or is there something going wrong in my RNA extraction/cDNA synthesis?
You should try a No Template Control to determine if you're seeing primer self-amplification or DNA contamination. You can also check the melt curves/band sizes of your sample and no-RT control products to see if they are the same species.
when water amplifies well it is likely that you have post pcr product contamination of one of yor reagents or in your working area/pipettes. If only some primers amplify it suggests that the contamination originally came from a sample where only those primers worked. run an agarose gel to see the size of your amplimers.
Cathal Dold
Signals in No-RT control may be due to the causes below;
1. Contamination of reaction components with target sequence.
Possible solution: Use separate work areas for mastermix preparation and template addition.
2.Primer-Dimer Formation
Solution: Reduce primer concentration and Perform melt-curve analysis to determine if primer-dimers are present.
3. Amplification of Genomic DNA (in RT-qPCR experiments): If the PCR primers for the cDNA template are able to anneal to the genomic copy of the same gene, an amplification signal may be observed in the no-RT controls.
Solution: Treat RNA samples with DNase enzyme before reverse transcription.
- Badges
- Science method