In my RT-qPCR the same set of primers gives different Ct values in the whole cell extract for wt and mutant strain in budding yeast. The difference is more than 5 cycles, up to no signal at all. The same whole cell extract looks well with primers for other genes (the difference between wt and mutant is lees than 0.2 cycles).
Am I right to expect similar Ct values for same primers? If yes, what is going wrong in my reaction?
Thanks, Anastasiya.
Dear Anastasia,
have you checked the efficiency of your primers (by serial dilution of a sample i.e. 1:10 Ct difference would be around 3.3 Ct)? I would assume the primer pair is not well designed and you have problems at the beginning of the amplification?
Best Soenke
Dear Sonke,
What do you mean by problems at the beginning of amplification? Another thing is that these primers seem to work perfectly well before.
Thanks, Anastasiya.
How do you know this isn't a genuine result? If you're getting similar values for other genes, and a 30+fold difference for this one gene, perhaps that's telling you this gene is affected by your mutation?
Dear Anastasia,
I'm now trying to answer a third time, the last two once were always followed by a log out during writing the message.
May be I got some thing wrong in my first answer, specially when you now wrote that the primers worked perfectly prior.
I'm also not sure if John isn't right. When you are performing a ChIP, I would assume that the binding of all other primer pairs (which does not really change) are not affected by the mutation. When you now perform a ChIP the protein you are detecting is maybe not binding any more to the sequence you are looking with the primers. If your input (non ChIP DNA) is still giving the same Ct and than the Chip-DNA drops down in the mutant the protein you are IPing might not bind any more. That might also be detected by a reduction of the transcription of the subsequent coding protein in the cDNA.
I hope that helps
Best Soenke
Dear Anastasiya,
assuming that:
- by ChIP-RTqPCR, you mean ChIP-qPCR and you are trying to see if your protein of interest binds to specific regions in the genome, since RT-qPCR refers to evaluation of gene expression
- WCE is "whole cell extract" or in other words "input" and represents the purified genomic DNA without IP
- you are using the very same WCE as input for your IPs
...this sounds very strange.
Questions/Suggestions:
- Do you measure your chromatin/DNA concentration before ChIP or are you using the same number of cells for your IPs? I usually use 50 µg of chromatin per IP (depending on the antibody and the target).
- Since the SAME WCE works well with other primers, it seems to be a problem of the primers themselves. The region you are amplifying is not in the mutated portion of the genome, right?
- Have you looked at the melting curves? Any particularities there?
- As already suggested: Have you evaluated the primer efficiency?
Let me know, what is happening, because I'm genuinely interested =)
All the best!
Kevin
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