I started with 5s as an internal control. I have extracted total RNA by different methods including TRIzol Ls and Qiagen RNA extraction kit. I also tried 3 different input volume of RNA in DNase treatment. Different cDNA dilutions have been tested in real time PCR but I still get high ct values. I may not use miRNAs as control due to their probable functions in pathogenesis of the disease I am working on. Does any one have any idea what to choose as internal control in serum samplas?
I don't understand why it would NOT be ok to use any variety of stabley expressed housekeeping genes that don't sit in your pathway (gapdh, b-actin, b2m, hprt, etc) you can try a few and if they are similar between samples you know have been generated from a similar starting biomass of material, than you can feel confident in finding an internal control.
Hi Elham
In case of body fluids we still don’t have a consensus reference for normalizing lncRNAs level, but as already suggested by Craig you can choose any of the stably expressed housekeeping genes (e.g., GAPDH, β Actin, etc.). Here are few suggestions which might be helpful:
1) Endogenous control: The overall function of an endogenous control is to serves as normalizer so you can choose around 4-5 endogenous controls and run the real-time followed by comparing the Ct values across all the samples, the endogenous control having the consistent Ct value across all the samples is the one with which you can proceed in your study.
2) RNA concentration: Well ideally 1 µg of RNA is sufficient for cDNA synthesis, or I would say even 500 ng is enough until your gene has a very low expression level which I think is happening in your case because you are working with serum samples. The RNA quantity in serum samples is often very low, and another important thing is their stability/ half-life as they degrade soon. I would recommend you to carefully isolate your serum sample and immediately snap freeze it if you want to use it later post isolation you need to amplify your RNA which will improve your Ct values.
3) Endogenous Poly A tail: Some of the lncRNAs have poly A tail while few don’t so to make sure that you are not losing any of your samples, please polyadenylate all the lncRNAs before proceeding towards cDNA synthesis.
Good Luck
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