Hi all,
I am performing QPCR with the E-cadherin gene and getting ct values around 11-12. I had done the same experiment two years back but back then the ct values ranged around 25-26. Can anybody explain what's happening?
Are ct values around 11-12 for a gene like ECAD acceptable?
Thanks in advance,
Swarnali
Hi Swarnali,
Have you checked the amplification curve? Also check the expression of your GOI from Gene expression Atlas website, so the expression maybe changes depending on several factors.
Lastly and the most likely cause is using high concentration of cDNA, so again control the amount of cDNA you used before and now.
Best...
Hi Swarnali Kar
In addition to what Mohamed Khashan suggested (assuming you do a two step process ie reverse transcription and PCR), it is a good practice to have your Housekeeping gene aka endogenous control along with Reverse transcription control, Non targeted control (NTC) and an Inter-assay control in your PCR plate to troubleshoot.
1) Check if you have loaded the correct amount of cDNA as Mohamed Khashan suggested
2) Check if your reverse transcription (RT) blank control is negative, if the RT blank comes positive, it means you have problem in reverse transcription.
3) Then check if your NTC is negative, if it is positive, then the PCR primer mixture is contaminated
4) Check if your House keeping gene is expressed properly example GAPDH, PPIA etc.,. change in house keeping gene suggests problem with cDNA
5) lastly use a known cell line with a known target for inter assay control ie., every time you do a PCR, add one sample with same target for every plate. For example, irrespective of what your gene of interest is, add cDNA from T47D cells targeting PPIA every time. You should only use this to compare your data between two PCR plates that you did on a different day or session. if there is a difference in the Ct values of IAC, the latest PCR reaction is wrong and there may be a contamination, then refer to above steps.
If you still have problems I suggest you extract fresh RNA again from the cell line with a standardized number of cells. (for example 1 million cells rather than a T175 flask of cells), Keep an eye on the freeze thaw cycles as well.
Hope this helps!
Thank you Ritihaas Surya Challapalli and Mohamed Khashan for your reply. I will first standardize cDNA concentration as you suggested.
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