Initially, one year ago, I was using the 5 micromolar concentration of primers and 100 ng of cDNA input. At that time, I was getting the ct values for GAPDH around 18. Now, I used different concentrations of primers (100ng, 200ng, 500 ng, 1 micromolar, 5 micromolar and 10 micromolar). But I am getting same ct values approx 35 for GAPDH. Even I tried it with different input of cDNA (50 ng and 100ng). But I am getting the same results.
And the rt pcr cycle configuration is as following:
10 min hold at 95C, Stage 1: 95C for 15 sec, stage 2: 60C for 15 sec, stage 3: 72C for 20 sec.
So, I would like to ask you what could troubleshoot for this situation.
If someone has any suggestions.
Please do let me know.
I appreciate any help you can provide.
Thank you
It is possible that the answer lies with the cDNA production. You could try borrowing a sample from another researcher and run it in your RTPCR
If your PCR chemistry is optimized and free of inhibitors, Ct is mostly influenced by the starting abundance of your target. If you used 100ng input for cDNA synthesis, if you kit allows more (i.e. 400 ng), then you can try to use more input for cDNA synthesis. Remember, if with 100 ng input you are getting 35 Ct, then doubling your cDNA input from 100ng to 200ng would roughly give you 34 Ct, likewise going from 100ng to 400ng would give you roughly 33 Ct. If you went from 100ng input to 50ng, what used to give 35ct would be 36 Ct now. Remember PCR is essentially a doubling reaction, so the more you give it the more it will produce of whatever template you gave it (assuming reaction is optimized).
Is it the same cDNA from a year ago? If that is the case it has probably started to degrade, especially if it has been frozen and thawed. My vote would be to make new cDNA and try again (and check the kit expiration date). If it's new cDNA, see if anyone else (who uses the same reagents and machine you use) is having the same issue. Perhaps one of the reagents has expired or the machine needs calibration.
The most likely explanation is the cDNA, but you never know. One time I had very poor results due to the machine needing cleaning (someone else spilled their plate and didn't wipe it up). Another time the bulb burned out.