5 replies to this topic

[susanna]

Hi,

I'm extracting RNA from quite old (2 years) and small FFPE slides, for gene expression analysis on qPCR.
So i first want to check my RNA quality before performing qPCR since RNA quality has a profound impact on the results.
Does anyone performed/know some assays to determine RNA quality?
Thanks in advance

greetz

[hannama]

If you have access to a Bioanalyser machine, they are excellent for checking RNA quality. I think you can also run formaldehyde agarose gels but am not certain as I never had to do it myself.

As a general point, see if you can find any papers referring to RNA extraction from your organism of interest; they usually include an image of the RNA run out on a gel (so if yours looks like theirs, it's probably ok) and describe how they assessed the quality of their RNA.

Hope this helps,

hannama

[Trof]

Agilent bioanalyzer if you have it.

If you don't (like me), then try denaturing gel electrophoresis, we prepare it like this:
normal 1% agarose gel with EtBr in it
Load with:
3 ul running buffer (same as for electrophoresis, we use 0.5x TBE)
2 ul 6x loading buffer (contains 30% glycerol and 0.05% bromphenol blue or/and other Elfo dyes)
1.8 ul formamide
1 ul RNA

And look for two bands of 28S and 18S, which should have ratio around 2. More about it (with pictures) in Ambion note.

Edited by Trof, 30 September 2010 - 07:41 AM.

[susanna]

and the nanodrop? Can that one be used too?

Trof, on 30 September 2010 - 07:40 AM, said:

Agilent bioanalyzer if you have it.

If you don't (like me), then try denaturing gel electrophoresis, we prepare it like this:
normal 1% agarose gel with EtBr in it
Load with:
3 ul running buffer (same as for electrophoresis, we use 0.5x TBE)
2 ul 6x loading buffer (contains 30% glycerol and 0.05% bromphenol blue or/and other Elfo dyes)
1.8 ul formamide
1 ul RNA

And look for two bands of 28S and 18S, which should have ratio around 2. More about it (with pictures) in Ambion note.

[Trof]

Nanodrop only tells you how many nucleotides you have in your samples and how pure it is. It doesn't tell anything about their condition, if they are degraded or not.

[f9120096]

why not try to check the OD280 260?
and reextracted RNA from your extracted RNA.
I am a junior student, hope this simple words would work.