To Fizban - miRNA (Mar/29/2009 )
FOR miRNA microRNA ISOLATION SEE THIS PAPER:
MicroRNA isolation and stability in stored RNA samples.
Mraz M, Malinova K, Mayer J, Pospisilova S.
Biochem Biophys Res Commun. 2009 Dec 4;390(1):1-4. Epub 2009 Sep 19. Review.
-Gaetanodiitaly-
Fizban on Oct 16 2009, 11:02 AM said:
EmilyG on Oct 15 2009, 06:50 PM said:
Hi Fizban
I've been trying to isolate miRNAs from human serum using TRI reagent BD with glyco blue to enhance the yield which appears to be working. However, the peak I get on the nanodrop appears at 270nm, which I think is TRI/Trizol contamination.
Have you seen a similar thing with your samples?
Have you got any suggestions for how to get rid of it?
Help much appreciated, tight time scale and little money to get pilot data for a bigger grant!!
BW
Emily
P.S. I will be using the miRNA on microarry chips as the downstream application
I've been trying to isolate miRNAs from human serum using TRI reagent BD with glyco blue to enhance the yield which appears to be working. However, the peak I get on the nanodrop appears at 270nm, which I think is TRI/Trizol contamination.
Have you seen a similar thing with your samples?
Have you got any suggestions for how to get rid of it?
Help much appreciated, tight time scale and little money to get pilot data for a bigger grant!!
BW
Emily
P.S. I will be using the miRNA on microarry chips as the downstream application
Hi Emily,
i'm sorry but i have bad news 4 u. You WONT get rid of it! The 270nm peak is quite recurrent when u try TRIzol extraction but is not isolated to that method. You'll find it also using miRVANA PARIS kit and other RNA extraction kit.
I've spent some months trying to get rid of it but with no luck.
only thing i havent done is looking at my RNA with microfluidic electrophoresis (with Agilent for ex.).
Up to now i extract my RNA and test it with miR17 to check if i have comparable Cts among my samples.
Hope it helps
Fizban
Never saw this peak myself. Have been using miRVANA PARIS. Wouldn't this peak greatly distort your 260/280 ratio?
-Baars01-
You sure you're not confusing this with a peak at 230 nm?
-Baars01-
newmirnaman on Mon Mar 30 02:15:01 2009 said:
Hi Fizban
You are one of the most active member in this forum now, and you mentioned that you use trizol method to isolate the miRNA, which worked very well.
Would you mind to share your protocol with me? could you forward a copy of your protocol in detail to me?
this is my mail. newmircorna@gmail.com
Thanks a million in advance.
Hi Fizban,
Thanks for your very valuable contributions towards our researches.
Yes the trouble I have been facing is that I tried to extract RNA last week from PBMCs from whole blood including virus in it. It was given to me as some kind of supernatant and they asked me to centrfige , get rid of supernatant which contains a lot of virus in it and add the trireagent to the pellet and follow the rest of the protocol. I did everything properly but got no RNA. I was wondering what did I do wrong? normally I always use Trizol for cell lines and it works perfectly well. By the way I want to use the RNA for miRNA validation and quantificiation.
Thanks very much.
cheers
-findik44-