RNA extraction - (Dec/01/2006 )

Hi
Do we have to use "freshly prepared 70% ALCOHOL" while doing RNA extraction? I did an expet. with about 1.5 million cells X 4 samples and all four gave very low RNA content. Thinking back-I used 70% alcohol prepared 3 weeks back. Could that be the problem?
Thanks!

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What protocol you used for the extraction?? I don't think that you need to prepare fresh the alcohol. I don't think that's the problem unless you had used an alcohol that everybody uses. You should use a dilution for that purpose only. Is super important that you use only rnase free items/reagents and all the materials and equipment is for RNA extraction use only. If you are using a column for the extraction check that is the right one for the quantity of cell,because it could get clog and the yield will be low. Hope this help.

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Thanks a lot! That was helpful. I use the RNeasy mini protocol.
Jaya

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i use 80% ethanol and once i made it like 50 ml aliquote , i use it until finish

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you don't need to yous fresh 70%ethanol as the interesting property is just 70%.
By fresh ethanol, it seems it may mean that it's for minimizing contamination (just by the fact an old slution may have been used several times, and despite the fact it should be a dedicated material, air contamination may occur.
So a dedicated RNA solution, "old from 3weeks" is ok.

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Prepare my 70% EtOH and store it in -20C or 4C. Prefer the former than latter if stored for a long period coz EtOH is a little hygroscopic and also have high tendency to evaporate as well, resulting in lower % of EtOH if stored long at RT.

Agree with everyone that 70% EtOH should not cause RNA degradation, unless RNase contaminated H2O was used to dilute the absolute EtOH when preparing it.

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Rneasy protocol shouldn't give you much trouble. As I told you before check that you are using the correct size for the quantity of cells, that the centrifugation is at the correct rpm/g. The step before the elution make sure that membrene is dry (centrifuge for a 1min or 2). The last step I advice that add the half elution buffer wait 5min, centrifuge, add the last half wait again 5 min and centrifuge. Good luck

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When I culture cells to do RNA extraction at 48 hrs, is it OK to add fresh media in between (say, around 24 hrs.?) Or, should I just leave it like that? When I did the experiment, I added media at 24 hrs . but there was difference of opinion between 2 staff -it is ok vs. not ok. Any thoughts?

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We use to make the 70% ethanol and store it at -20 degree C without aliquoting it.

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hi all,
do u have that protocol for RNA extraction through coulmn.
if yes, then plz. send the desired links.
thanks
puja

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