TRIzol reagent protocol (Life Technologies, Inc., Gaithersburg, MD)

[sobored]

I used TRIzol reagent protocol (Life Technologies, Inc., Gaithersburg, MD) for isolation of my human tumour total RNA and i wonder if there are any kits i can use to isolate mRNA from this suspension? I have looked in Qiagen and they only have kits for mRNA isolation from animal tissues and not human suspension.



THanks for any help.

 

[Moonbear]

Humans are animals.

I'm not entirely sure what step you're stuck at. The protocol that comes with Trizol is pretty good and straightforward.

 

[sobored]

I am looking for a kit to extract mRNA from this total RNA suspension. The Quiagen kits are only for extracting mRNA directly from the tissues and not from total RNA suspension.

 

[Moonbear]

Do you mean something like this?

http://www.superarray.com/newsletter/RNA.html

Enriched mRNA samples are also suitable for microarray gene expression profiling experiments. SuperArray recently developed the ArrayGrade mRNA Purification Kit that combines the oligo-dT affinity purification technique with a magnetic microparticle technology. The Oligo-dT14 is immobilized on 1 micro, super-paramagentic microparticles. The oligo-dT microparticles are uniform, colloidally stable and non-porous spheres that offer high surface area and can remain in suspension to facilitate the interaction between the poly (A) tail and oligo-dT.

 

[sobored]

Thanks for that link.

Since you have read the protocol of Trizol. Do you know why i should incubate my RNA sample for 10 minutes at 55 to 60°C? This would degrade my RNA and how come i use it for further working?

The protocol says; "5. REDISSOLVING THE RNA
At the end of the procedure, briefly dry the RNA pellet (air-dry or vacuum-dry for 5-10 minutes). Do not dry the RNA by centrifugation under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Partially dissolved RNA samples have an A260/280 ratio < 1.6. Dissolve RNA in RNase-free water or 0.5% SDS solution by passing the solution a few times through a pipette tip, and incubating for 10 minutes at 55 to 60°C. (Avoid SDS when RNA will be used in subsequent enzymatic reactions.) RNA can also be redissolved in 100% formamide (deionized) and stored at -70°C."

http://www.invitrogen.com/content/sfs/manuals/15596026.pdf

Thanks.

 

[Moonbear]

As far as I know, that's just to get it into solution faster.

 

[iansmith]

The step at 55C is to get the RNA into solution faster. It is the same idea has with DNA. You will not see a significant degradation of RNA if you RNA was carefully resuspended with any material that is RNase free.

You may skip this step if you see that your RNA went into solution quickly after you added your dilutent.

 

[sobored]

The protocol says that i have to air-dry the isolated RNA. Would not this contaminate the sample with RNase and damage my RNA sample?


Thanks.

 

[iansmith]

Don't air dry the tube right side up. Tube upside down or on its side will reduce the odds of contamination.