Protocol Online logo
Top : Forum Archives: : Real-Time PCR

Question concerning quality RNA - (Apr/24/2008 )

Hi everyone,
I obtained the RNA quantification results with Experion (see below photo). In the graph, it seems no 'important degradation' (no big furious peak or hill before 18S peak), but the ratio 28S/18S is rather low (1.27). Is my RNA really degraded ? If NOT, why did I obtained a low ratio 28S/18S ?

I repeated (5-6 times) the RNA extraction of my samples which have stored at -80oC after being frozen by liquid Nitrogen at collecting. But I always have low ratio 28S/18S, except the first time (RNA extraction of the fresh tissues just after harvesting). However, I used the same protocol (RNeasy Plant Mini Kit -QIAGEN) every time. I have 2 other questions, please:
1/ According to many protocols, the frozen samples (ex: plant leaves treated by liquid N2) may be stored at -80oC for several months for RNA extraction. Is it right ? Is there any problem of RNA degradation during storing ?
2/ Someone told me that the homolysates (in lysis buffer) stored at -80oC overnight before continuing next steps of extraction would give better quality of RNA. Does anyone have experience with that protocol ?
Thanks for any suggestion or explication.

-thanhtunghuynh-

QUOTE (thanhtunghuynh @ Apr 25 2008, 04:23 AM)
Hi everyone,
I obtained the RNA quantification results with Experion (see below photo). In the graph, it seems no 'important degradation' (no big furious peak or hill before 18S peak), but the ratio 28S/18S is rather low (1.27). Is my RNA really degraded ? If NOT, why did I obtained a low ratio 28S/18S ?

I repeated (5-6 times) the RNA extraction of my samples which have stored at -80oC after being frozen by liquid Nitrogen at collecting. But I always have low ratio 28S/18S, except the first time (RNA extraction of the fresh tissues just after harvesting). However, I used the same protocol (RNeasy Plant Mini Kit -QIAGEN) every time. I have 2 other questions, please:
1/ According to many protocols, the frozen samples (ex: plant leaves treated by liquid N2) may be stored at -80oC for several months for RNA extraction. Is it right ? Is there any problem of RNA degradation during storing ?
2/ Someone told me that the homolysates (in lysis buffer) stored at -80oC overnight before continuing next steps of extraction would give better quality of RNA. Does anyone have experience with that protocol ?
Thanks for any suggestion or explication.


From Flieg and Pfaffl RNA integrity and the effect on the real-time qRT-PCR performance, 2006, Molecular Aspects of Medicine, 28s/18s is not a good indicator of RNA integrity. RIN is, if you can get access to/afford an ABI Bioanalyser 2100. Thankfully the previous paper also provides evidence that 18s expression (analysed through qPCR) correlates highly with the RIN that the bioanalyser gives.

I make sure that I use the same amount of RNA (measured by a Nanodrop after DNase treatment) for RT, and find 18s expression with ~0.5 Ct delta between all samples. Anything greater than that I discard and resynthesis the cDNA for, or failing that make special pains to scrutinise the results for.

Of course, with the current understanding and pace of qPCR this could all be invalidated tomorrow, and you should check it in your own system (mine is retina and brain during development and after injury in the rat).

Cheers,
Maset.

-maset-