Hi all,
I am performing qPCR on Trizol extracted RNA from BMDMs stimulated with LPS. I am probing for TNF-alpha cytokine production as well as a few other candidates. My TNF qPCR results yielded reasonable Cq values ranging from 21-27 depending on my treatment condition. My GAPDH values however ranged from 37 - undetectable (40 cycles). I went ahead and did a standard PCR to determine if my GAPDH primers were a problem and obtained an amplicon at the correct size (35 cycles, Phusion PCR).
Any thoughts on why my GAPDH qPCR is failing? I used 100 ng of RNA for cDNA prep (Superscript III First Strand Synthesis). And followed by using 2 uL directly from cDNA reaction for qPCR (Dynamo HS Sybr Green).
Would be happy to provide other details!
What is your primer efficiency? My guess is either your transcript is very low abundance or your primers are not efficient enough to use in qPCR and you are getting poor amplification of your target gene.
Thanks Katie,
I am not sure if I would be able to calculate the primer efficiency since even my undiluted samples yield Cq > 35. These are primers that have been used in previous publications on BMDMs so I am confident that they should work (and the standard PCR confirmed that they can amplify my target from my cDNA). I am leaning towards the issue being the transcript being in low quantities. My starting RNA was not of the best quality (A260/230 ~ 1.3-1.5).
In house we have a Cq cut off point of 36, so I would probably be very cautious of a Cq of 35. If you are using Trizol, you could have phenol carryover, phenol is extremely inhibitory to polymerase, so you need to make sure that you get rid of as much as possible. This normally shows up in the A260/A280 ratio as the phenol ring will increase the A280. So I always recommend two or three chloroform washes after the Trizol, this may improve the quality of your RNA.
You can use another reference genes like18S rRNA, Actin, ubincutin, Tublin.. and so on. Of course, you should take into account the RNA quality particularly A260/230 ratio higher than 1.5, which means a low amount of the phenolic and protein inhibitory to the polymerase.
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