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How to check RNA quality


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5 replies to this topic

#1 susanna

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Posted 30 September 2010 - 06:44 AM

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

#2 hannama

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Posted 30 September 2010 - 06:50 AM

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

#3 Trof

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Posted 30 September 2010 - 07:40 AM

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.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#4 susanna

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Posted 01 October 2010 - 07:42 AM

and the nanodrop? Can that one be used too?


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.



#5 Trof

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Posted 01 October 2010 - 07:51 AM

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.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#6 f9120096

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Posted 12 October 2010 - 12:43 AM

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.




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