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Loss of RNA conc. and quality in Qiagen RNeasy Protocol


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#1 KMM73

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Posted 02 June 2011 - 11:55 AM

I performed the Qiagen RNeasy extraction through to dissolving the pellet (I dissolved it in 30ul of RNAse free water) then quantified using the NanoDrop. My results were high concentrations of RNA and high 260/280 ratios but low 260/230 ratios. My lab was running low on spin columns so I froze my samples and purified them a week later following the rest of the protocol, picking up where I'd left off. The result was very low concentrations of RNA as well as very low 260/280 and 260/230 ratios. I can not figure out where I am losing RNA. Before beginning the second half of the protocol, I allowed my samples to fully liquefy on ice and pipetted up and down to be sure that the pellet was fully dissolved. Does anyone know what the problem might be?

#2 KMM73

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Posted 02 June 2011 - 12:01 PM

PS The samples I extracted from are sponges in Trizol.

#3 Trof

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Posted 03 June 2011 - 06:02 AM

I'm not sure I follow it, if you disolved the pelet and measured RNA concentration, that's the end of the protocol. So what other steps are you talking about? Can you clarify?

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#4 KMM73

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Posted 03 June 2011 - 06:58 AM

Sure, thank you for your reply. In my protocol, the first half is the basic extraction of RNA from Trizol samples. After obtaining a pellet, it can be dissolved and quantified, stored, or one can continue on to the second half of the protocol which involves further purification through the use of several reagents and silica membrane spin columns. After I redissolved the pellet and quantified the RNA, I had high concentrations of RNA (~1000-1700ng/ul) as well as high 260/280 ratios but very low 260/230 ratios, indicating that further purification was necessary. I stored my samples in the -80C freezer until the spin columns arrived about a week later. I then thawed my samples on ice and proceeded through the rest of the protocol involving the addition of Buffer RLT and ethanol then transferring to the spin columns, etc. After completing the purification steps, I quantified the RNA again and expected high concentrations and high 260/280 and 260/230 ratios. However, the concentrations were much lower (<50ng/ul) and both ratios were very low. My reagents were no more than a couple days old and my samples had been carefully thawed on ice to prevent degradation. I have been researching the issue but have not found any answers. I simply do not understand why I am losing such large amounts of RNA. I have never had this issue when I perform the entire protocol without storage.

#5 Trof

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Posted 03 June 2011 - 07:23 AM

OK, first I found your method very strange, isolation from Trizol following the protocol is a complete RNA isolation with all necessary nucleic acid purification. There is no need to put this to a column and purify again. The RTL is a lysis buffer and the column kit is designed for cells as a starting material not purified RNA, so who knows what's going on there apart from normal on-column loss.

Second, RNA isolated from Trizol can and usually is contaminated with DNA (we try to minimise this by taking only upper 2/3 of the aqueous phase after phase separation) that can give you false higher spectrophotometric read.

If you need clean RNA I recommend to isolate from Trizol and do DNase treatment with Ambion's Turbo DNA Free.

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





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