RNA Kit vs Trizol - (Jul/14/2007 )

I'm wondering if anyone could tell me what the difference is I am seeing between my total RNA samples. The difference is seen between the Sample I extracted using a kit (Lanes 2, 4 and 5) and the samples I extract using the Trizol method (Lane 3 and 6). The kit sample shows a large band in every case (Lane 2, 4 and 5) which is not seen in the Trizol method (Lane 3 and 6). Is this DNA contamination? Also the Trizol method shows more faint bands towards the end of the gel. The kit uses glass fiber matrix columns to filter out the RNA. It says it effectively isolates RNA >200bps. Would the Trizol mehtod be more sensitive at getting lower MolecularWeight RNA than the kit (Lane 3 and 6)?

http://www.biology-online.org/biology-foru...load.php?id=936 <- gel image

-CIBL-

QUOTE (CIBL @ Jul 14 2007, 04:32 PM)

im working a little bit with RNA and im going to tell you just my opinion.
RNA isolation with trizol is better than any kit.
the most kits have a great scale of DNA contamination and i think that that is the band you ve got on the top.
i suggest work with trizol.
check you RNAs for DNA contamination. put up a pcr (without reverse trans). if you have the expected size product, you ve got a contamination.
I cannot answer about the small molecular weight RNA but generally i do not trust the kits for RNA. RNA is avery difficult molecule in my opinion an kits dont work very well. hope i helped a little...

-Tasos-

Thanks man.. It looks like the Trizol is better at isolating the small RNa.

-CIBL-

QUOTE

Also the Trizol method shows more faint bands towards the end of the gel. The kit uses glass fiber matrix columns to filter out the RNA. It says it effectively isolates RNA >200bps. Would the Trizol mehtod be more sensitive at getting lower MolecularWeight RNA than the kit (Lane 3 and 6)?

By using Trizol you can isolate 5s RNA which is smaller than 200 bases. But kits usally can't isolate such small fragments. Moreover as my friend say in kit you may face with DNA contamination problem, however it can be solved by applying DNase treatment. But I think you can obtain a larger scale of RNA by using kits while comparing with trizol. By the way there are commercially available kits which are designed for isolating small RNAs such as miRNAs. In our lab we use this kit and it works.

-gothica-

QUOTE

Yeh... We got the kits because they were over 50% off.. THe kits yield more RNA and they are alot faster and Easier. Thanks for the Advice guys. I'm going to have to try both I guess and see what I come up with..

thanks.

-CIBL-

I don`t think so ,if it is DNA contamination problem,the different bands always be a smear .But in your picture the band is clear and order-bounded.You are suppose to take it into account .

-redmission-

column cut off are too big for any small RNA. There is a recent paper from abrasimov, in which they add glycogen and precipitated the flow through of the column. So they get an enriched small RNA fraction.
Your gel shows 2 major band which i assume are ribosomal RNA. The usually admitted 2:1 ratio 23/16 or 28/18S is not very clear. So i guess you got some RNA degradation.

Let me aks : the "top" of te gel is at the BOTTOM of the image ,isn't it ?
it seems that "bands" you see are the wells
i did that mistake once.... so i just ask.

-fred_33-

I use both. Trizol for pre-adipocytes and the RNA Isolation Kit (Promega) for everything else. I have more RNA with the Kit. And I also prefer them more than Trizol, because of the handling. It fits me better.

-Sumpf-