Hi All,
Im a newbie to real-time pcr. My task is to optimise a real-time gene expression assay for two GOIs expressed in mouse brain astrocytes. I had quantified cDNA after RT-PCR using Qubit fluorometer (Invitrogen) with their ssDNA assay kit. Has anyone used Qubit to qnantify cDNA? my first assay was done using 1ng, 5ng and 10ng of cDNA, and was able to see amplification using all concentrations of template. However, the internal control used was beta-actin, which had a higher Ct than one GOI. does this mean i have to select another internal control for my assay? how can i figure which is the best internal control, will i have to try out many? i have used oligo dT primers for RT-PCR, so can i use 18s rRNA as internal control, since it doesnt have a poly A tail? is it necessary that the internal control should always have a lower Ct than GOIs? and also one of the GOIs have a Ct of 36, can this be used in the analysis, or will i have to ignore it and understand that it was not expressed? is there an optimum Ct range for which we can use the delta-delta Ct method?
sorry for bothering you with so many questions, but i would really appreciate if any one could please help me by giving an opinion.
thanks in advance.
Thank you Wang for that reply, it gives a lot more confidence in what Im doing.
What level of Ct you get with your reference gene? Also, don't use 18s if you use oligo dT. I understand that by doing the reaction with different template amounts you wanted to check primer efficiency? If yes, then I think 3 concentrations is not so much, you should span a range of 0.01 to 50 ng of template. Remember that your reaction efficiency needs to be close to 100% to get reliable results. If it's not, re-desing primers.
Thanks Kris, the Ct for my reference gene is 22 and that of GOI is 16. I am using pre-designed primer probes from ABI, so do i need to check efficiency?
if they are validated by them, then you don't, if they are only pre-designed, not validated then it's a good idea to check them anyway. The cts you have, seem to be fine to me. Since you said that the other GOI is 36 ct, you can try to use higher concentration of the template (a ct of 14-15 with the other GOI would still be fine). I'm wondering, if you have any amplification in the water of no-rt control samples?
Hi Linda, I agree with Kris and I am also wondering at which Ct your water control or your no-RT sample is coming up. If these are still coming up at around 38-39 I would suggest to use more µL of your template: each duplication of the amount (for example from 1 to 4 µL will be two duplications) is giving you 1 Ct value earlier and therefore more accurate as well as increasing the difference between you background (in water or no-RT sample) and your "real" amplification. Good luck!
I understand, that you are talking about a one-step RT-qPCR? You only add your RNA and incubate in one reaction tube?
If yes, then obviously you have to quantify only RNA, you can't really quantify cDNA.
Of course, you can do that. Just remember, that you RT is never 100% efficient, also the efficiency between samples may vary, so it may happen that you actually have differing amounts of cDNA going into your qPCR. It shouldn't be a big problem since you always have a reference gene on a plate.
unless, that is, you're doing an absolute quantification based on a standard curve.
Thanks a lot Kris, your final remarks actually answered my initial doubts about quantification. I'm enlightened!
For example if you extract RNA from cells, there is often some residual genomic DNA in your sample. The genomic DNA contributes very little fluorescence to your overall signal if you use the RNA specific Qubit dye, so you can quantify only the RNA (which presumably is what you're interested in); similarly, if you run a 1st strand cDNA synthesis, you likely have RNA in your sample, but you can estimate the amount of DNA resulting from your cDNA synthesis using a DNA specific Qubit dye
To eliminate DNA contamination you can also do DNase treatment after your RNA isolation before the cDNA synthesis (cheaper then specific kits). Furthermore, if you design your primers over exon-intron boundaries and use a short elongation time you will not be able to amplify genomic DNA, only RNA, since the introns of the DNA are too long to get amplified in just 20-30 seconds.
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