High Ct values in ref gene as well as target gene - (Oct/25/2013 )
I did taqman assays for the cDNAs from mouse skin biopsies. There were six different groups containing 5 animals each.
We isolated RNA from all the skin biopsies individually and synthesised the cDNA after DNA clearance (3µg RNA used).
When I analysed the data, for one group alone in all the 5 mice, the Ct values of both HPRT (internal control) and target gene were very high in the range of 35-40. For all the other groups the Ct values of HPRT was around 25-30 and the target gene around 30-35.
The RNA concentrations were good. In agarose gel, I could see the two RNA subunit bands.
What is the problem here? How can I lower the Ct values in this particular group?
Thanks in advance.
Sounds like your DNA preparation was not optimal. I would try to make new cDNA from any remaining RNA you may have. I have had huge fluctuations with my HPRT1 values when my RT reaction went hay wire. HPRT1 Ct values of 25-30 makes it almost impossible to quantify your data.
Try your HPRT1 primers on another sample that you know has worked perfect in the past. Maybe freeze-thaw of your primers has caused degradation, but I doubt it. Also run each HPRT1 reaction on a gel and see if you see sharp bands.
Thanks a lot for your reply. I did the cDNA synthesis similarly for all the 30 samples. But the samples from only one group (all 5 mice) out of the 6 grps had this high Ct in HPRT and Target gene.
"HPRT1 Ct values of 25-30 makes it almost impossible to quantify your data" - If I use more RNA for cDNA synthesis will it improve?
"Try your HPRT1 primers on another sample that you know has worked perfect in the past" - I always include a cDNA which worked fine previously in my RTPCRs. It also had a Ct of around 25.
"Maybe freeze-thaw of your primers has caused degradation" - I used fresh aliquots of the primers.
I would keep the amount of RNA you use for cDNA synthesis constant. It shouldn't matter as long as you only analyze your data from the controls run with those increased RNA concentrations, but it would make it difficult to quantify results from previous experiments (i.e. checking expression of a gene from this experiment, to a previous experiment) - does that make sense?
It is possible that your RNA has degraded over time. Is there anyway you can get fresh DNA?
Try another reference gene and see if you get higher than normal Ct values?