Since we wanted to know, if a particular gene is expressed in the fly antenna, we did a RT PCR (as we don't have a very good antibody to show it in the immuno or western) and ran a conventional DNA gel and there is an expression of our gene of interest in the fly antenna. However I have been asked for qPCR. We are not comparing our gene of interest in different conditions to show a relative expression. The idea was just to show that this gene is normally expressed in the fly antenna. So how will I do the qPCR calculation just to show that there is an expression of our gene of interest?
End-point qPCR it is called. A "Yes" or "No" answer in other words.
Isolate RNA from the antennae and either perform RT first to make your cDNA (be sure to DNase treat your RNA before RT so you don't end up amplifying gDNA during the qPCR) and run the qPCR with an appropriate mastermix depending on whether you are adding DNase-treated RNA to the qPCR or cDNA to the qPCR. Using a sample mixture, perform a serial 1:3 dilution for 15 points (all in duplicate or triplicate replicates) and calculate the exponential amplification efficiency of your plot of Cq vs log of dilution by: Eamp = 10^(-1/m) -- do this on the most log-linear/useful/valid region of the plot without eliminating too many points on either end to attain that. The high concentration end and low concentration ends of the dilution profile can have common problems of inhibition and/or primer dimer contribution, respectively. "m" in the above equation is the slope of this chosen line.
Based on what you find your good relative dilution standard curve range to be, dilute all samples to the same spot (same ng/uL concentration) within that sweet range of that standard curve, and run all samples individually in triplicate replicates at the same singular concentration within the range of your curve (preferably within the earlier part of the curve - to get the strongest signals possible).
Run the qPCR for at least 40 or 45 cycles. If using a SYBR Green-based mastermix, be sure to check melting curves for product fidelity. The qPCR, if it amplifies something specifically, note the cycle that the signal(s) cross threshold (quantification cycle; Cq) and use the formula: Relative quantity = Eamp^(b-Cq) to compute each relative quantity. "b" is the y-intercept of the relative dilution standard curve mentioned above ("Eamp" is as been described above).
Ideally the dilution curve and your individual samples would be run at the same time - but not absolutely necessary. Another reason to perform the dilution curve is to prove that your primers (or primers-probe) are actually working in the first place, and that they are working in such a way that they indeed respond to sample dilution in a reasonable way. Be absolutely finicky about primer design beforehand, check, re-check, and triple-check that they are absolutely correct for the target you have in mind.
You can actually just stop at the point of gathering Cq values. If they show up before 40 cycles, and are specific for your target, you have your answer that the mRNA was indeed present. Be sure to not get false positives by only amplifying gDNA. This is a simplistic answer but probably more than answers your question?
Your question has one concern... you said it was a DNA gel... if you only proved that the gene was there (e.g. just did PCR on gDNA), that does not prove that mRNA was expressed for this target - it just shows that the gene is present in the genome of the fly you are studying. For qPCR, use primers that span a genomic intron-exon junction to be doubly sure that gDNA is not giving you your signal.
Hi Jack, Thank you very much for your reply. I really appreciate your help and the detailed explanation. May be I was not clear with my question. Actually I extracted the RNA from the antenna, made a cDNA and amplified my gene of interest from the cDNA with the specific primer (exon-exon bridging) and ran a DNA gel. Thus I showed it on the gel that my gene of interest is expressed in the antenna. However one of my reviewer asked for qPCR and I did not know the proper qPCR calculation showing just gene expression (not the fold increase). May be I will do normalization with house keeping gene. thanks
Ahh - that makes more sense now. If this is a small-scale experiment, be sure to use at least 2 or 3 reference (housekeeping) genes for normalization as then you will be abiding by the current 'best-practices' advice out there. However, it seems redundant that you should have to show this by qPCR when you've proven the expression of your transcript already by the conventional method? If all you want from the qPCR is to show existence of expression of your transcript and are not interested in fold increase or decrease (e.g., differences in transcript level from sample to sample), then it seems there is really no need for you to do the qPCR (unless your advisor merely wishes you to learn qPCR?)...
Only for yes/no, the conventional PCR is enough.
Maybe the reviewer wants to have a hint about how strong is the expression when compared to a reference gene.
Additionally you can use serial dilutions of a cDNA clone of your gene of interest and run together with your samples. This will allow you to calculate the copy number of your gene of interest in that sample.
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