Hi y'all,
I am running into some difficulties with qPCR (SYBR). To give y'all a brief background summary, I transfected some cells (ASO) and am running qPCR to see its effect on known genes using qRT-PCR. The first time I ran a qPCR, I saw little to no amplification. I re-ran it again and this time I went back to the beginning (RT-PCR from the original RNA) and was able to get some data. However, I am running another qRT-PCR but this time it's not working at all, as in I am seeing zero amplification, which is weird because, again, we know for a fact that some of the genes are supposed to show up in abundance. I was very careful throughout the whole process, so I don't think there was any human error. I haven't run a gel yet, so that's something that I have in my mind. I just wanted to see if y'all have faced similar problems.
Thanks in advance!
SYBR green is very sensitive to freeze-thaw cycles. Has yours been in and out of the freezer a lot?
Also, how was your RNA quality/quantity that you started with?
If you are encountering problems with your qRT-PCR, consider employing this troubleshooting process to identify and address potential problems (see the attached image). There are several factors that may contribute to the difficulties you are facing, and this systematic approach is intended to help you identify and resolve them effectively
Hi,
Are you sure about your RNA quality after isolation? I advice you to run a qRT-PCR by targeting well-known regions by using commerical primers or high quality primers that you already have. If you know your RNA quality and sure about your protocol, you may consider changing your primers. You also need to be sure about cDNA synthesis and its final quality.
Good luck!
Hi!
I would suggest that you check the following points:
1. The quality of your RNA and cDNA
2. The primers you are using. Have you tested them before? if you did a self-design, there is likely a problem, which is why no amplification is showing. As you said, you are certain about the existence of some genes. Try using primers used before in your lab for the same genes or primers already published in papers targeting genes you are looking for.
3. Also are you using a housekeeping gene (B-actin or GAPDH), what about the amplification of those two. If also no amplification so the problem is from the extraction quality. If there is an amplification of HKG so the problem is from th primers used.
Good luck !
I agree with the suggestions above. Also, is anyone else having the same issue and if so, what are any commonalities? Same SYBR? Same primers? Same QPCR machine?
My best guess would be your cDNA is not good. But, I did have a QPCR machine itself stop working one time (and I was the only user at the time). Double check your program too (sample setup and cycle parameters).
- Badges
- Science topic