Hello All.
I am not entirely sure if what I am seeing is a problem but I wanted to get it out there and see if there this is a problem which I will need to resolve.
I am doing a lot of qPCR's at the moment and I have noticed my housekeeper is coming off, on average, at cycle 20-22; may I ask if this is an expected value for a housekeeper gene? I am only slightly worried because I have seen other works in which the housekeeper is coming off much earlier at cycle 15. However my housekeeper is being detected before my target genes so I don't really have a worry about whether the housekeeper is suitable for this assay; I'm only slightly worried if maybe the cDNA reaction is not wholly synthesising enough cDNA.
In summary, I am extracting RNA from animal tissues using a Qiagen kit and I quantify the RNA using a nanodrop; these are usually quite high e.g. 1200ng/ul, 1000ng/ul, 980ng/ul e.t.c but I think this is because I add a 10 minute incubation time when I apply the elution buffer to the spin column and I elute in fairly small amounts (70ul). I then make 1ug of cDNA from my RNA and add 5ul to the PCR reaction. So if I am correct: 1000ng in 20ul cDNA reaction = 50ng/ul. 5ul of this = 250ng of cDNA. 250ng of cDNA in a 20ul PCR reaction = 12.5ng/ul. Is this a good amount of cDNA to use?
Looking forward to hearing your thoughts!
Cheers,
Adam
Extremely sorry, I forgot to mention that we dilute our cDNA 1:100 after the reaction. so in fact I'm left with 0.125ng/ul rather than 12.5ng/ul that I stated above.
Thanks again.
Hello Adam,
This doesn't seem to be a problem. If your housekeeping gene's CTs are not deviating much across experiments then you are fine.
Thank you all very much for your informative and reassuring answers, it is extremely kind.
My housekeepers Ct replicates do not vary much, definitely less than 0.5 cycles, therefore I will carry on with this protocol.
Thank you all once again.
If your housekeeping (reference) gene was 18S RNA, the 0.125 ng cDNA/uL qPCReaction would give you a lower (lower than 20-22) Cq value here. So, you are obviously not using 18S as the HKG, forensically speaking.
Just out of curiosity, what is your housekeeping (AKA: reference) gene here?
Your Ct values look fine to me! I would echo the response by others that the Ct values would be quite dependent on the housekeeping gene used. We use a low-medium abundance reference gene and will sometimes get Ct values of 25-30!
Your Ct values are Ok! For a HK gene such as Actin beta and Gapdh 1 microL of concentrated cDNA (standar reaction 1000 ng/20 microL RXN) will give Ct arround 19, depending on the cell type or tissue. If I'm correct you used 1:20 of such a reaction.
Good or bad depend of the reference gene you are using and the expression values of the genes you want to detect. You should try to evaluate the expression levels of usually known as "reference genes" with high level expression as GAPDH, ACTB (Ct values around 15-20), medium expressed genes as SDHA, RPL13A (22-27) and low level expressed genes TBP, HMBS (27-30). Then you will sure that you would be able to detect the expression of genes in any relative expression level.
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