Elham Taheri Bajgan

Still there is no consistent internal control to normalize serum gene expression(including lncRNA).

My advice is while isolating RNA added two or three synthetic RNA.

Design primers also these three synthetic RNA.

Perform qpcr and for normalization use average of three synthetic RNA as a internal control.

Best wishes

Sincerely

Prabu

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

@Tarun pant

Thanks Tarun

Actually I have tried concentrating serum RNA using NORGEN RNA concentration and clean up kit. It did improve ct values of my internal control as well as the other transcripts I was working on. I highly recommend that specially for RNAs from body fluids.

about the polyadenylation, I cant use it since the lncRNA I am working has multiple splice variants. Some of them lack a poly A tail. So I rather not to lose them in detection prosedure.