RNA extraction

[prn0907]

Dear All,

I have tried extracting RNA with various kits, however the yield was quite low....about 20-40 ng/microlit in a volume of 30 microlit. Is this acceptable? How can I increase the yield?

The yield from Trizol method was excellent, but the purity wasn't great. It was less than 1.6. Therefore, I would like to ask if the DNAse treatment for Trizol method would be helpful to get rid of genomic DNA?

Regards,
PM

[mchlbrmn]

We couldn't know if the yield is low without knowing the number of cells, or amount of tissue used.
Do you mean 1.6 as the A260/A280 O.D. ratio? I have found that the guanidinium extraction gave me lower OD ratios than column purification, but it usually worked in most downstream applications. I think a 1.6 ratio was common for me.
DNAse treatment of RNA will lower the amount of genomic DNA, and I usually did do this before reverse transcription. However, often traces of genomic DNA can still remain, so it is best to try to remove the genomic in the RNA purification (although I'm not sure if genomic is always distinguished from precursor RNA?).
Genomic DNA is not distinguished from RNA by OD since both DNA and RNA absorb at A260.

[relaxin]

You can clean up your RNA after Trizol method by using RNeasy spin columns. There is an on-filter DNase digestion step. Your high yield from the Trizol method may be due to DNA contamination.

[Daigoro]

Hi all!
I also have a question regarding Trizol RNA extraction. When you vortex your sample in Trizol, can it cause RNA degradation? I tried 2 methods for homogenizing my sample (filamentous fungi) in Trizol. The first one is the multi-vortex genie and the second is by simple vortexing. I discovered that multi-vortex gave a higher yield of RNA than the other method. But I was not sure whether it was due to RNA degradation or better lysis.
Thank you!

[Daigoro]

You can clean up your RNA after Trizol method by using RNeasy spin columns. There is an on-filter DNase digestion step. Your high yield from the Trizol method may be due to DNA contamination.

What if we use LiCl to reprecipitate RNA sample from ethanol precipitation? LiCl usually does not precipitate DNA efficiently.

[relaxin]

Hi all!
I also have a question regarding Trizol RNA extraction. When you vortex your sample in Trizol, can it cause RNA degradation? I tried 2 methods for homogenizing my sample (filamentous fungi) in Trizol. The first one is the multi-vortex genie and the second is by simple vortexing. I discovered that multi-vortex gave a higher yield of RNA than the other method. But I was not sure whether it was due to RNA degradation or better lysis.
Thank you!

I do not think vortexing sample in Trizol can cause RNA degradation. The reverse will be true. Slow lysis may lead to RNA degradation. It may be helpful if you use electric homogenizer such as Polytron.

RNeasy spin column cleanup will remove contaminants from Trizol. If you do not think DNA contamination is not a problem, you can skip on-filter DNase digestion.

[molecular_micro]

Hi,

With regards to vortexing in the presence of Trizol....This is not a problem. Many of the methods I have used incorporate mechanical disruption with bead beating devices/cell disruptors in the presence of trizol. Trizol will 'protect' your sample (it can be used as a storage medium for RNA). Mechanical disruption w/trizol should also increase your yield quite drastically. With regards to column clean-ups; My organism tends to have carry-over 230 contamination (causing low 260/230 ratios) even after on-column clean-up. You can overcome this by EtOH precipitating overnight, followed by a long spin the next morning, 75% EtOH wash and re-suspension in molecular H2O.