I am struggling with make RNA probe for in situ hybridization for a while. I wanted to know what is the best efficient way for extracting RNA right after RNA labeling reaction. The protocol that I have said use G50 column but some people extract the RNA via phenol/chloroform. Is there anyone that have experience using both of them? which prevents loosing RNA?
Hello Zahra,
Both of these two methods will have certain degree of RNA lost during purification. The use of G50 column is to get rid of probe fragments during synthesis. Because those fragments may increase the background(reduce probe specificity) during ISH. I don't think phenol/chloroform purification of RNA can have the same effect as using G50 column for probe purification. Good luck!
Cheers,
Philip
Hi Philip,
Thanks a lot for your answer. I could not end up by making RNA after a couple of times try so I though it might be possible that I loose the majority of my RNA in G50 purification step.
You can try this one
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/Bulletin/1/11274015001bul.pdf
Btw, did you check the concentration of RNA after in vitro transcription?
Thanks Philip! The G50 that we use in my lab is from GE. I think all are size exclusive and basically have similar function.
Yes, I nanodrop the result after RNA labeling reaction, and unfortunately the graph was so spiky and messy which means there was not any RNA on it.
Hi Zahra,
Did you look at what your RNA looks like before you purify it?
Phenol/Chloroform is a bit tricky with all the phase separation and all. Usually, I just use LithiumChloride to precipitate the RNA after the transcription reaction. I have summarized my protocol and the trouble shooting here:
https://insitutech.wordpress.com/2016/01/11/how-to-make-riboprobes-for-in-situ-hybridization/
https://insitutech.wordpress.com/2016/09/26/troubleshooting-riboprobes-preparation/
I hope this helps.
Good luck!
Simone
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