Hello, I'm researching gene expression of cells after some treatment, which cause apoptosis. when I compare 2 different population of cells (WT and crispr KO of some gene), I get the same Ct for my gene of interest, but I have higher Ct in GAPDH. I tried using some other acceptable housekeeping genes, but I get the same result with them.
can I rely on these measurments and conclude that there is an increase in the expression of my gene of interest?
If I understood it right, the Ct of your GADPH after treatment is higher. Usually GADPH is used to normalize your gene of interest, and it's better to try at least 3 housekeeping genes in case one of them is somehow regulated by your treatment, and eventually using as fold-increase the mean of the fold-increase of your gene of interest normalized with the two remaining housekeeping genes. Generally you should be able to normalize the gene of interest with your housekeeping genes of choice, and this is to avoid calculation mistakes related to pipetting errors. Now you say that your treatment causes apoptosis. Simply, can it be that the increase of your housekeeping genes' Ct is just caused by cell death and eventually fewer template? If so, try readjusting your experimental design so that you start the qPCR with approximately the same number of cells.
thanks. well, the thing is that i load the same amount of rna into the reverse transcriptase reaction. but still, 3 housekeeping genes with distinct functions are with higher ct. i guess that this is due to some rna degradation during apoptosis, but the question is if i can assume that the same degradation is happening to my gene of interest and calculate fold increaaed based on th ..
At this point I would say yes, but I would cross - check this with the RIN (RNA integrity number, which is basically an index of the degradation of your ribosomal RNA and therefore of RNA in general).
Shalom Moshe,
I agree with Davide, it might be best to keep the number of cells you use for the extraction same in both groups. I think that if you tried 3 different house-keeping genes, you can trust them more than just comparing Ct for your gene in different samples. You always need to compare relative levels of expression.
To me the fact that you see higher Ct for 3 different genes indicates that you load extract from fewer cells in your treated group (even if total RNA amount you load is the same). What also might be happening is that the global transcription rate in your apoptotic/treated cells is affected or you observe some RNA degradation in your treated cells. If your gene of interest was upregulated by your treatment however, even after general RNA degradation there should be still more transcripts left and that might explain why you see that tendency. Especially if you see that repeatedly.
You might want to collect the same number of living cells before RNA extraction and then add the same volume of RNA (not concentration/amount of RNA) of the RNA to the reverse trancription reaction and then see if you still see the same. Especially given that you will be probably repeating the experiment anyway (you don't want to make conclusions based on 1 experiment only).
Best of luck,
Michal
Is the primer set you are using for the gene of interest a proven primer set? Could it be measuring some part of the sequence that is not affected by your treatment? If it was reading "junk," that neither increases nor decreases by your treatment, that could be the reason you see an increase in the proven housekeeping genes but not your target... Just a thought.
I have similar problem occured. I also seeded the same number of sells and treated compoundn at different concentrations. After extraction, adjusted RNA amount was used for rt-pcr to make cDNA.interestingly, Ct value of housekeeping gene was increased in the dose dependent manner. To confirm it, I tested with different housekeeping genes including 18s, rpl32, GAPDH and HPRT. In addition, tested again with different cell type (3T3- L1). Still Ct values are increasing. I am sure it is not due to mistake in the process of study, since other experiments with other compounds is fine. I gusess it maybe due to Apoptosis. If it is due to the apotosis, how I can confirm the apotosis without housekeeping gene. Should I use other test rather than realtime pcr? I wonder how Moshe solved this issue?
I am having the same issue here. I have used GAPDH, SDHA, B2M and 18S. All of these HKGs are showing varying Ct values.
1. I am starting with diff. cell numbers but am using the same RNA amount for the cDNA synthesis.
2. I am isolating RNA from a monolayer and the supernatant. The GAPDH and 18S Ct values for the monolayer are almost identical. There is less deviation for the supernatant 18S CT values. I have used GAPDH for WB with the same treatment and same cell line and it showed to be a good HKG.
the cells I am collecting in the supernatant is a mixture of live and dead cells. Could it be that my HKGs are being degraded causing high Ct values?
Is there a good HKG for dying cells?
Is there a way of separating live and dead cells from the supernatant?
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