Having problem with primers for qPCR - (Nov/30/2013 )
Hi everybody, recently I have a serious probem with the primers I have designed for qPCR gene expression studies. I already have the GAPDH primers so in the beginning I have started with designing the primers for three different genes. The workflow I did is as follows:
1. I looked up the transcript sequences from NCBI Gene
2. I have designed the primers spanning exon-exon junctions (Tm~60C 20 bases)
3. I have checked the primers from NCBI Primer BLAST according to the RefSeq mRNA database
4. I have checked the stability and Delta G values for hairpin, self dimer and heterodimer from IDT Oligo Analyzer
Finally I ordered them after I saw everything is clear.
However, I only got product from the GAPDH primers when I run them on agarose gel. So I decided to order new primers which belong only to one exon (not spanning another). And the result was again negative, I only got weak bands on agarose gel in 2 of 3 primers (and quite good band of GAPDH product).
Just to mention, I have used random primers for cDNA synthesis.
Here is the question, where is the mistake or misunderstanding that I could be wrong in this experiment?
From the info you provided, you are doing quite right. It is rare that such designed primers did not give you any amplification. There are two possibilities: one is the expression of the gene is quite low; 2nd is that your RNA may be degraded, although GAPDH still gave you band.
Let me ask you some questions: How many cycles did you use for GAPDH and your target gene? How much RNA did you use for RT? Are you testing the expression in cell lines and how many lines or RNA samples have you included in the experiments?
Answers to those questions will be useful for troubleshooting. In the meantime, you can run a gel using your RNA to check its integrity. You can find the method here:
Thank you very much for your reply pcrman. There are actually three different genes as eNOS3, VEGFA and ANGPT1. I thought in the same way as you did, the expression of those genes can be low. I got visible but weak bands for VEGFA and eNOS3 but nothing for ANGPT1. The band of GAPDH was very strong and clean though.
I used the same input of RNA for all the primers (as 50ng) and made the PCR reaction with 30 cycles according to the kit's protocol. The material is rat tissue and I am planning to observe the expression differences of the genes between the case and control.
I will try to run the RNA samples on a gel and also run a pcr with increased primer input next. Hope the problem will be solved then...
You can test your non-spanning primers using genomic DNA instead of cDNA as a template. If they don't work there, then they definitely won't work in an RT reaction.
Great idea, thanks. I will try gDNA as well. Hope this helps to understand.