polyA+ yields from total RNA?

[pmiguel]

What yields do you typically get of polyA+ RNA from a given amount of total RNA?

We use the Ambion PolyA Purist Mag kit and cannot seem to get 0.1-0.5% yields after 2 cycles of purification. (1 cycle leaves too much rRNA around for sequencing.)

If we can get people to give us several hundred ug of total RNA, then 0.1% yields are acceptable. But often asking for that much RNA is not realistic.

Advice?

--
Phillip

[AdamB]

I did a two-round poly(A) pull-down (same kit) last week and got about 2-3% yield.

[pmiguel]

I did a two-round poly(A) pull-down (same kit) last week and got about 2-3% yield.

What tissue/organism was the RNA from?

Thanks for your response,

Phillip

[shurjo]

I get around 150 nanograms of poly(A) from 10 micrograms of very fresh LCL total RNA using two rounds of DynaBeads oligo(dT) purfication. Ribosomal contamination is around 100,000 reads per 15 million.

[AdamB]

What tissue/organism was the RNA from?

Thanks for your response,

Phillip

That was from rat cardiac myocytes.

[scooter]

I would agree with jamessmith01. 2-3% is my typically yield after two rounds. Overall length of the tail can certainly influence yield, though I would not expect the rat cells to be particularly short.

I have had very good luck with the Ambion kit. To me it is much better than Oligo-tex from Qiagen.

[pmiguel]

I would agree with jamessmith01. 2-3% is my typically yield after two rounds. Overall length of the tail can certainly influence yield, though I would not expect the rat cells to be particularly short.

I have had very good luck with the Ambion kit. To me it is much better than Oligo-tex from Qiagen.

How long do you do the hybridization for? I am wondering if degradation during the oligodT hybridization is limiting our yields. We use Ambion magnetic beads, but I notice that incubations are much shorter for the Invitrogen (Dynabeads) kit.

We rarely do mammalian work -- most of our samples are from plants or fungi, although we do some fish and insect work as well.

--
Phillip

[scooter]

I have not used the mag bead format and have only used the oligo-dT cellulose version, which I have found to be quite robust. I cannot remember off the top of my head how long we incubate, but I think it is no more than one hour, then wash, elute and rebind to a fresh set of cellulose beads.

I have a feeling the Mag beads result in less yield. I can check with my friend at Ambion who developed the purist kit and get back to you.

[AdamB]

We're also using the cellulose kit, not sure if that was clear or not.

[pmiguel]

I have not used the mag bead format and have only used the oligo-dT cellulose version, which I have found to be quite robust. I cannot remember off the top of my head how long we incubate, but I think it is no more than one hour, then wash, elute and rebind to a fresh set of cellulose beads.

I have a feeling the Mag beads result in less yield. I can check with my friend at Ambion who developed the purist kit and get back to you.

Please do. I avoided cellulose because I used to hear people complaining about how terrible the process of isolating polyA+ RNA was. But this was in the early 1990's so the situation will likely have improved.

Also, though our yields seem low, it is always hard to be sure that the issue is not just that the relative abundance of polyA+ RNA is low in the samples we get.

--
Phillip

[scooter]

My buddy says that the originally vetted % recovery using the mag bead version after two rounds would be ~1%, so it sounds like you are in the neighborhood. My experience tells me that the cellulose version should give you more like ~2% after two rounds. This is for samples like Human Brain Reference RNA, so admittedly a fairly rich source of polyA+ RNA. You are certainly correct that your particular samples may have less.

As far as cellulose beads go, I had also heard the rumors back in the day. However, I have been pretty happy with the purist kit; especially since recently I did a direct comparison with oligo-tex and the latter was quite bad (as least the BA trace looked very strange for oligo-tex but the purist samples had a very nice smooth distribution and no detectable rRNA peaks).