I am trying to optimize my primers for qPCR using SYBR green chemistry. To do this, I pooled 10 of my mouse samples (control and experimental groups), and performed serial dilutions of 1:4 for a total of 7 Standards. I used these to create a standard curve and calculate primer efficiency. I obtained the sequences from Origene's website, so I know that the design of the primers is not the issue. GAPDH had an efficiency of 97 %, while IL6 had 134%, TNFa had 167%, and MCP1 had 126%. I know my dilutions aren't the issue either, because the GAPDH efficiency wouldn't be so good. I've tried diluting my cDNA at multiple conentrations (1:1, 1:4, 1:8 and 1:32), and 1:1 yielded the best results. I'm at a loss of how to make this work. Any help would be greatly appreciated!
Hi Sarah,
With this RT-qPCR are you just wanting to get standard curves from each gene to validate that the efficiency is within 90-110% If yes, seeing that IL6, TNFa, and MCP1 are over the designated range, how do the melt curves look? If the melt curves are not ideal there might be contamination, carry over RNA-DNA, or you could be experience Polymerase Inhibition
Shawn Krueger
Hi,
Yeah, I'm just trying to validate that the efficiency is within 90-110% so that I can rely on them to draw accurate conclusions when it comes to running my actual experiments. The melt curves look fine for each, showing only one product. So, I'm not sure if it's maybe an annealing temperature issue, or what. My first thought was polymerase inhibition, which is why I ran dilutions of my cDNA, and my least diluted yielded the best efficiencies. Thanks for your help!
Sarah Elizabeth Eysoldt
So the fact that the efficiency is improving only with the diluted samples indicates that you might have contamination. I would check your RNA on a nondenaturing gel to see if that looks okay before troubleshooting the next step. If it is contamination somewhere throughout the process I usually start by checking the RNA quality first, because if you have sheared RNA for use in Reverse transcription you could be carrying over some unwanted stuff. Did you run a no template control for the RT-qPCR?
Shawn Krueger
Sorry, maybe I wasn't clear! my 1:1 dilution of cDNA yielded better efficiencies than my cDNA that was more diluted. That was one of the first things I tried troubleshooting. I ran a no template control for each target, and there wasn't any contamination.
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