Hello everyone,
I work with C57BL/6 mice and administer exercise to them to process PCR in muscle tissue. However, I have not yet found a suitable housekeeping gene. We tried GAPDH, HPRT1, 36B4, RLP19, and α-Actin, but they did not work properly. Do you have any suggestions for other genes besides TBP1 and B2M?
Have a good day.
At this point, if you have already tried with GAPDH as a housekeeping gene for your RT-PCR, I strongly suggest switching to 18S rRNA. However, keep in mind that 18S is a very strong housekeeping gene. To achieve accurate quantification of your target gene expression levels, you will need to dilute the cDNA template for 18S, usually at a ratio of 1:100 or even 1:1000.
Good luck!
Hello Parham Farahvachi,
Thank you so much for the information. May I as another question. I obtained the cDNAs from 100ng RNA and these cDNAs worked in RT-qPCR without any dilution. Therefore I feel like their concentration is not so high, do you think, still I should dilute them before using 18SrRNA?
Thank you very much in advance.
Hi Merve Çulha,
Cheers, it's always important to help each other. Speaking on your second question, you're right. You have to diluite your cDNA for 18S if you want to use it as housekeeping for your RT-PCR. You have always remember that in contrast to GAPDH or B-actin, 18S being a ribosomal component has a high expression levels in comparison to many other genes. If you don't diluite it, you risk to wrongfully quantify your interested genes. Based on my experience, if you have retro-transcribed 100ng of RNA extracted from tissues and the qualities is good, I recommende watering down your cDNA 1:1000. However, if you want to reuse a cDNA stored at -20 for more than 3/4 months diluite it at a ratio of 1:100.
- Badges
- Science method