I am measuring the gene expression of my target gene DDX43. I took 2.5 ul of cDNA for Q-PCR. I am using SYBR GREEN for my q-PCR. The concentrations of RNA for most samples were 20-60 ng/ul.
The CT value for housekeeping gene hypoxanthine phosphoribosyl transferase 1 (HPRT1) is around 32-36 and CT value for for DDX43 with all samples were"undetermined". When I search in expression atlas for my target gene. I find it below cutoff in the tissue I used for RNA extraction.
Can I add up to 5ul of my samples in running q-PCR? or this result is consistent with below cutoff expression which means it is undetected so undetermined in qPCR?
"undetermined" means your samples didn't amplify. You can increase the volume of your template in your reactions.
@Katie A Burnette I want to ask if I need to increase the volume of the template why the house keeping gene amplify and the target gene didn't?
@Priyankana Banerjee
Thanks for your answer but how the house keeping gene amplify and the target gene didn't? Can this occurs due to high template concentration?
https://www.researchgate.net/profile/Katie-Burnette
I want to ask if I need to increase the volume of the template why the house keeping gene amplify and the target gene didn't?
Hi Noha,
Yes you can raise the template amount providing you will do the same for the housekeeping protein.
I would like also to suggest that you check your primer of interest and to make sure that there are no bubbles in platee wells before carrying out PCR.
Good luck
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