4 replies to this topic

[alqga]

Hello,

I am extracting RNA from different tissues (liver, spleen, brain) and also of isolated immune cells from head kidney and blood with the Trizol method. With liver and spleen I got really good 260/280 and 260/230 ratios but when I extract RNA from the brain and from the isolated cells I got good 260/280 ratios but very low 260/230. I have performed the process at the same time so I don't know why there are such huge differences between tissues. I read that in some tissues is just more difficult to extract pure RNA, so my second question is, how important is this 260/230 ratio for qPCR?

Thanks!

[memari]

Do not waste your time for 260/280 and 260/230 ratios.

Just read the OD for 260/280.  Then devide 1Microgram/Microgram of each sample. For example if the OD is
0.303,  devide 1/0.303= 3.3.
This mean that you take 3.3 micro liter of the sample for RT-PCR and then one tenth of this cDNA for QPCR.

nevertheless, you can improve the ratios by these steps:
1- keep cells in TRIZOL for 20 min without chloroform.
2- add more chloroform to TRIZOL.
3- Vortex vials horizontally and not vertically on the Vortex for 10 seconds.
4- also add a step with just chloroform plus vortex after chloroform:TRIZOL.

-----
Babak

Edited by Curtis, 31 August 2012 - 05:30 AM.

[alqga]

Thanks for answering! So it means that a very low 260/230 ratio won't affect my qPCR results?

The thing is that I have to compare gene expression among tissues, that's what i am a bit worried about such differences. Anyway I have the reference gene so I guess that if there is something wrong with the samples I will be able to see it in that qPCR...

[memari]

Yes
The most important thing is normalization.
Read the OD of all samples.
Decide how much RNA you want for RT-PCR.
This kit below need  100 Femto gram to 1 microgram.
For example you want to take 1 microgram from all samples.
so devide  1/each sample.
for example the ODs are  0.3, 0.45. you devide one to them
that is
1/0.3= 3.3 Microliter   ,   1/045= 2.2 microliter
http://www.bio-rad.c...A-Synthesis-Kit

[memari]

this link has very good info

http://www.invitroge...tating-rna.html

https://www.ncbi.nlm.../pubmed/9067025

Edited by memari, 29 August 2012 - 09:34 PM.