4 question for real time PCR - (Oct/06/2010 )

HI everyone:
I’m going to evaluate expression level of CaBP-D9K&CaBP-D28k mRNA in rumen and duodenum by real-time PCR (SYBR Green I), but I got some question to be clear since I am new in Real Time PCR……

1: I get a little confused in choosing Absolute Quantification or Relative Quantification. I just want to compare gene expression level in different tissue……

2: Which method of Relative Quantification is better, relative stand curve or delta-delta Ct method?

3:Must I make a standard curve if I choose the delta-delta Ct method? Is this method can be trust (I mean amplification efficiency of target gene& reference gene may not the same)?

4:If I have to make a stand curve, Should I must clone target gene and reference gene into plasmid for build a standard template?

Sorry for my poor English and many questions. I would be really grateful to have any suggestions from you guys, Thanks.

1) If you compare expression level of the same gene(s) in different tissues and not the absolute number of transcripts, you want to do Relative quantification. You just need to find a good reference gene for that.

2) Delta-delta Ct method is still most popular, but it doesn't account for different PCR efficiencies of your target and reference genes. More accurate is Pfaffl method (efficiency corrected delta-delta Ct)
This tutorial covers pluses and minuses of all three methods.

3) Delta-delta Ct requires no standard curve, Pffafl method needs efficiency computed from a single standard curve (done in replicates for better precision) and standard-curve method needs standard curve in every run

4) In relative quantification you don't need plasmid standards, you just dilute the cDNA (so you better chose one with abundant expression of your genes) both for standard curve method and Pffafl efficiency.

Good pages to read about qPCR.

Thanks Trof for your kindly reply.

Hi all, I m also doing real time. For question no 4. what if my cDNA did not express the gene of interest?

for my understanding, to do delta-delta ct method, first we have to do an efficiency test for both target and housekeeping gene (preferably from the same sample of cDNA). To do the efficiency test, we need to construct standard curves for both genes. For my case, i m transfecting gene of interest into a cell, which mean my cells naturally did not express the target gene. My question is, how do i construct a standard curve for my target gene since my cells (control) did not express the gene of interest? can i replace with plasmid stardard curve for the efficienct test?

thank you inadvance.

What about your cells after transfection with your target gene? They should express your gene of interest then (hopefully).

because i'm doing nucleofection, i did not do selection for stable transfection. which mean my control sample is a mix population of cells. In the other word, they are expressing gene of interest, but not 100% of the cell expressing. is it alright for me to use this sample to construct a standard curve for my target gene?

thanks.