To Fizban - miRNA (Mar/29/2009 )

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.

Does glycogen is necessary for miRNA isolation by using Trizol?

newmirnaman on Mar 30 2009, 06:07 AM said:

Hi,

i saw your message and here i am. There's nothing strange in my trizol protocol.

I just followed the instructions and used my total RNA (my trizol is from invitrogen). at the beginning i used to do RNA extraction with PureLink miRNA isolation kit from invitrogen but then i saw that miRNA enrichment is not necessary.

If u have very small samples u can try using glycogen just before adding isopropanol. doubling the amount of isopropanol also helps sometimes.

That is all, no magic or whatever, simple TRIZOL.

Bye for now, contact me if u have doubts

Fizban

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

EmilyG on Oct 15 2009, 06:50 PM said:

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

Hi Fizban

Thank you very much for your reply, sounds like you have saved me a whole lot of work!

I have looked at my samples using an Agilent 2100 Bioanalyzer and saw nothing out of the ordinary - just nice low molecular weight peaks which should be the miRNAs. I'm just about to order a couple of TaqMan MicroRNA Assays to test out my samples and if that works well I think I'll just go ahead with the microarrays - no time to hang around!

Thanks again
BW
Emily

EmilyG on Oct 17 2009, 03:57 PM said:

Sounds good! Is Agilent 2100 able to tell u how much RNa do u have?
let me know about you results. if u wish we can compare protocols.
BW
Fizban

Hi Fizban

You can use the Bioanalyzer to measure concentration but this is compared to a ladder and is not as accurate as a nanodrop.
Unfortunately our ladder samples have been freeze/thawed a few too many times so the concentrations I get are always a bit lower than they actually are.

I have just found out you can get small RNA specific chips for the Bioanalyzer and I'm hoping to borrow one later this week to run my samples - I shall keep you posted about how these work.

I have not finalised my protocol for miRNA extraction from serum yet, got a couple more tweaks to try out before I'm really happy with it.

BW
Emily

Hi Fizban

I have just ran some samples on the small RNA specific chips for the bioanalyser and unfortunately the results were not great. I don't think they were sensitive enough to pick up the low RNA levels I have.

With more RNA the results could be quite useful as they give a detailed profile of your RNA according to size, with a % of miRNA present.

Out of interest, from what material are you extracting miRNAs from?

I'm using human serum samples and get around 200-400ng of RNA from 300ul. This is of course using a nanodrop reading with the slightly odd peak at 270nm so probably not completely accurate!!

My basic protocol is to use TRI reagent BD according to the manufacturers instructions with the addition of 1ul of glyco blue and 0.5M ammonium acetate with 1 volume of isopropanol to precipitate the RNA.
Does this sound similar to your protocol?

BW
Emily

EmilyG on Oct 22 2009, 12:45 PM said:

quite similar actually.
I use human plasma, 200-400ul of it. I use Trizol, add glycogen (1-2 ul) just before the 1ml of isopropanol precipitation. that's it. dunno about amount of RNA because nanodrop quantification is VERY far from being accurate.
that's it, seems very simple but worked very hard to set it.
Fiz