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Urgent - RNA extraction / RNase contamination ?

RNA extraction RNAse contamination

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4 replies to this topic

#1 Tabaluga

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Posted 15 March 2012 - 01:41 PM

Hi,

we extracted RNA from cells using the RNEasy Mini Kit (Qiagen) according to the protocol and eluted with 50 ul nuclease-free water. When I measured the RNA concentration (Nanodrop, absorbance at 260 nm) afterwards, it turned out to be extremely low (5 times lower than expected)). The 260/280 quotient was fine, however.

Afterwards, we realized that we might have mistakenly used normal autoclaved pipet tips instead of RNas-free ones, but only at a single step in the protocol.
Is it likely that the low concentration at the end could be due to RNase-contamination and subsequent RNA-degradation ? How strongly is RNA degraded ?

Thanks Posted Image

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#2 Curtis

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Posted 15 March 2012 - 05:56 PM

yes, it is usually low with mini kit, now worries. When I extract with Trizol I get like 3-5 times more than Qiagen mini kit. It is just the matter of what you want to do with the RNA after extraction. Of course the purity of mini is said to be higher than Trizol...though some people don't care about it much.

I don't think RNase effect is that critical as some textbooks and people warn you of it. Although I don't recomment this, I myself always extract RNA on my bench, without RNA Zap or RNase-away spray. I don't even use RNAse-free tips. Honestly, so far I haven't had any issue.....Trizol itself has RNase inhibition effect.

#3 Tabaluga

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Posted 17 March 2012 - 02:48 PM

Thanks for your reply. The concentration was 5x lower compared to the last extraction with the same cells (always using RNEasy Mini Kit), but so it's not likely to have been an RNase contamination... Hm, so the problem must be somewhere else....
By the way, I also always extract on the bench.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#4 helencollins560

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Posted 27 March 2012 - 11:26 AM

Tabaluga

I modified various other stages of the qiagen RNeasy mini plus kit to get good yields and RNA integrity.

I found that you actually need to put less tissue into the spin columns than qiagen states otherwise the tissue will clog the membrane and lower your yield. I went for 20mg of cardiac tissue rather than 30mg and that greatly improved my yield. Also, if you dont homogenize the tissue well enough this may reduce your yield. I kept all my tissues on dry ice to stop the samples from thawing and then homogenised with the Qiagen buffer. For cardiac tissue i used a good electrical homogeniser from fisher.
Your low yield could be due to the manner in which the sample was handled prior to use.

helen

#5 henryn

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Posted 14 November 2012 - 12:24 AM

Visit the link below to get more information on RNA Extraction.

Unless you are a sponsor of Protocol-online please don't advertise your stuff here without contributing anything useful! Bob.

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total rna





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