Hi everyone!
I extracted RNA from urine pellet using phenol-chloroform method. The yield of RNA was 160ng/uL. 3uL of RNA was used for cDNA synthesis in 20 uL reaction using Go-Script kit. (promega). Then 2uL of cDNA sample was used for qPCR using SYBR green for housekeeping gene. I did not get amplification. Could anyone help me please? Note: I run gel and RNA was a single band. I did not do clean-up.
If your RNA runs on a gel as a single band, then the problem is that your RNA is very degraded. What is a 'urine pellet' - I'm guessing that you centrifuged some urine? You might need to look at how your samples are collected and stored because RNA degrades very quickly. The best way to maximise RNA integrity is working fast and working cold. Is it possible to centrifuge the urine samples within minutes of collection, and then store them at -80C until processing?
Thank you so much Dr.Laura Leighton for your reply. I did extraction as much as quickly and maintained it in cold temperature throughout the work. I did not remove ethanol completely before dissolving the pellet because of RNA degradation. Is it a problem for getting one band?
Additionally, phenol carry over significantly decreases the PCR success. Be careful during RNA extraction. Try not to transfer trizol (or other brand reagent) to next step. I use zymo research extraction kit which is specially designed to be used for trizol based extraction.
Good luck
Laura and Kaan have already given some great answers, Have you checked that your Taq is amplifying anything at all? I've once had inactivated Taq polymerase. I'm sure there is only a slim chance of this happening but it has happened to me before. So worth checking.
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