Quick background: I want to compare the expression of two homologs in potato. The two genes are >80% identical, so designing primers has to be a tricky process. I have designed a few of my own, but I am getting super high CT values (30ish) while my HKG are amplifying around 15. My efficiency and standard curve for my HKGs looks good, so I know my RNA is fine, but the high CT values won't let me get an accurate standard curve for my two GOIs. This leads me to believe that I need better primers. All of the troubleshooting I can find on the interwebs points to using Primer3, but I can't use Primer3 because I need different primers for each homolog, and they need to be specific to each gene. So does anyone have any tips on designing qPCR primers.
The other option is to increase the starting amount of cDNA. So is it kosher to concentrate the cDNA reaction and use a dilution of it for my HKGs?
I am using bio-rad icycler and associated programs. cDNA was made using iScript reverse trasnscriptase supermix from total RNA extracted with the ... Any other details I might have missed, just ask.
Thank you so much in advance.
Yes the higher CT means lower expression from the other genes in comparison to your HKG. So try to modify the amount of the starting materials. Primer 3 can gave you many suggested primers spanning your genes so try it or do it manually for the non-homologous regions.
You can still use primer 3 and tell the program roughly where you want your primer and exclude the regions that are unsuitable. Even if you only have a very small region that primers can be specific, putting only this region into Primer3 will at least bring back good primers. You could try concentrating your cDNA but to bring the Ct value higher by just 3 cycles would need 10x more cDNA, and too much cDNA will inhibit the PCR reaction. Hopefully new primers will solve this for you.
Yeah, I was worried about the concentration of the cDNA part... I guess I'll just let Primer3 hammer away at it for a while, and see if I can't choose primers that are specific.
Maybe try QuantPrime, gives you gene specific primers, exon-exon covering etc
http://www.quantprime.de/
Hi, if I understand this correctly before design your specific primers you were having Ct of around 15 but now you have Ct of 30? Has the Ct values went higher for both forms? I normally work with teleost prologues from the 3R (normally >80%) and 4R (some times around 90-98% similarity). Sometimes what happen is that before you distinguish between paralogues you have 15 Ct but then discover that one of the is much less expressed and you have one that still up at 15Ct and the other goes to 30Ct (just an example). That is normal if the have gone to a process of subfunctionalization. Then, just concentrate the cDNA more, but think that the amplification is exponential, so, if with a sample diluted 1/100 times you get 30Ct, concentrating to 1/50 you will improve just 1Ct if lucky.
If both of your genes have increased the Ct to 30 that smell like a primer problem. It could be that your previous primers were binding other genes with similar regions and over estimating the abundance of your gene or that the new primers are forming hairpins, strong cross-dimmers, etc…
I have always design my primers using Primer 3 (20-22nt and with a Tm of 60oC) and then check them manually by align them to the original gene. Make sure they are truly specific, also, if for whatever reason your primers are design in the same region (with a 80% shouldn't happen, but you never know) just try to concentrate the differences between primers at the 3'-end. I also check for cross-dimmers and hairpins in my primers using NetPrimer (also free).
Hope that help.
Thanks everyone. I'll keep trying.
And Daniel: My CT didn't change, that's just the difference in CTs for my GOI (gene of interest) and my HKG (housekeeping genes)
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