miRNA extraction using Trizol - (Apr/25/2006 )

I am trying to extract miRNA using Trizol from cells. I read some threads already and it seems some of you are already doing this. I have some questions regarding the isopropanol precipitation step in the protocol: According to invitrogen to precipitate the small RNAs one should increase the amount of isopropanol from 0.5ml / ml of Trizol to 1mL / mL of trizol.

1. Should I stick to isopropanol or switch to ethanol precipitation?
2. If using ethanol, should I use glycogen in the ethanol precipitation?
3. Can I loose miRNAs during the 70% Ethanol wash? Should I modify this step? Maybe use 80% Ethanol?

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I followed the following protocol to extract total RNA (including the small RNAs) from cells:

1. Trizol followed by Ethanol precipitation (instead of isopropanol) with excess ethanol with glycogen (glyco blue from ambion)
2. Precipitation at -70C for couple of days (until I was ready to use the RNA).
3. 80% Ethanol wash.
4. Pellet dissolved in DEPC-H2O

Then I performed a DNase treatment for 15min at 37C then heat inactivation at 65C for 5min.

Then I performed RT-PCR on the samples using the AB miRNA RT-PCR kit for let7b and I compared this extraction to one performed using the Qiagen RNeasy kit modified protocol for RNA that includes the small RNAs (protocol on Qiagen website).

In the end only the Qiagen purfied sample gave a signal while the trizol did not. Is this because i somehow lost my small RNAs in the trizol purification? Or maybe there is too much salt leftover on the RNA I extracted using the trizol?

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hi
i wouldn't use ethanol precipitation due to the fact the amount of ethanol which should be used is quite high and i trust better isopropanol as it works at room temperature and etoh need cold temps.
even if uneeded, using glycogen is not distrubing further assays so for more secure, use it.

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I think isopropanol is work for precipitation of small RNA just followed the original protocol.
By the way it seems you neednot perform DNase treatment for the RNA used as template in ABI assay.

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According to Invitrogen you need to modify the protocol for trizol by adding extra isopropanol.

Anyways, I am in the process of comparing the following extractions for small RNAs:

Trizol with Ethanol and Glycogen

Qiagen RNeasy kit

Qiagen RNeasy kit - modified protocol for small RNA extraction (found on their website)

Ambion mirvana kit

maybe I should do an Trizol with isopropanol extraction too just to see.

BTW, does anyone know how much total RNA I should load into a 20% polyacrylamide gel (non-denaturing) in order to visualize small RNA bands?

I just want to state that the reason that I used ethanol over isopropanol was the fact that a colleague recommended it over isopropanol for miRNA extraction. He has alot of experience in the field (whereas I have none) so went ahead and followed his advice.

My main concern regarding the trizol protocol was the 70% EtoH wash. The 30% H2O content could mean loosing those small RNAs that are extremely soluble...

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I am going to purify some small RNAs using TRIzol and would like to know if there is any conclusion to what is the best methode for precipitation.
Should I use Isopropanol or Ethanol?

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you can do it with isopropanol first as it's the recommended procedure and works really good.
We have enhanced the trizol procedure with a colleague (he's now capable to do QPCR on 1cell) and we're using IprOH and glycogen.
For EtOH 70% wash, we resuspend the pellet, wait 10' on ice and then centrifuge.

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Hi,

concerning the isolations of small RNAs there was a recently published paper,
you can find the link below; I was just curious if anyone of you have similar experiences as in the paper or what your opinion would be about the instability?

I usually use Ambion but might switch to pure trizol protocol soon.
Cheers


http://www.ncbi.nlm.nih.gov/entrez/query.f...l=pubmed_docsum

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For me both the Trizol (with ethanol and glycogen ) and the ambion mirvana worked best.

The qiagen modified protocol does accomplish to purify small rnas, but the concentration is way too low.

I did not test the trizol with isopropanol. I just tought that both might do exactly the same; it is just a matter of personal taste.

There is also a small rna extraction kit from invitrogen -- which I haven't tested. Anyone have any experiences with this?

Invitrogen claims it is better (more small rnas gets extracted) than ambion's kit.

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I read your paper and I have a question: for the timepoint = 0 (i.e. the fresh RNA extraction). Was that ever frozen before assaying it with the qRT-PCR?

Also, for your triplicates: are they PCR-only triplicates (i.e. same cDNA assayed 3 times in the qPCR) or are they biological triplicates (i.e. similar tissue extracted 3 times, 3 different
rev. transcriptions, and different PCR for each)?

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