mRNA troubles

[dobbr493]

I was wondering what kind of quantities of total RNA have people used for extracting mRNA from? At our lower limits, we have 5ug, max usually 10ug. Is this a common amount to be using with success?

Also, having had trouble with the purification of mRNA, we are now looking at amplifying it to get the quantity we need. Has anyone done this with success from the quantities of total RNA above? Any comment on the potential bias introduced with the PCR and selection steps in terms of NGS results would be appreciated.

Thanks!

[chadn737]

That is more than enough, at least for Illumina. Last time I only had between 1-2 ug total RNA. The Truseq kits are supposed to be able to use as little as 100 ng.

[dobbr493]

Thanks for the reply.

Unfortunately we are not going down the Illumina path - gone for the 454 approach instead. The cDNA library preparation requires at least 200ng mRNA and we can't seem to get any with either the Roche magnetic bead kit or Ambion spin column kit.

[aliceb]

Have you had any luck?

I might worry less about the total RNA you have and more about how much mRNA you can squeeze out of it. I've had better results from the oligotex mRNA purification kit, but it might depend on your study organism. We've also had decent luck making 454 libraries with less than the required 200ng.

I'm in Auckland, so you can PM me if you have other questions....

[maiyosry29]

Hi aliceb,

I'm about to do RNASeq for mRNA samples using 454, but the problem is that I never reach 200 ng (the starting amount) !! when i saw your post I got very optimistic that u managed doing it with less amounts.......so how much mRNA did you use?

[maiyosry29]

Hi aliceb,

I'm about to do RNASeq for mRNA samples using 454, but the problem is that I never reach 200 ng (the starting amount) !! when i saw your post I got very optimistic that u managed doing it with less amounts.......so how much mRNA did you use?

[pmiguel]

The best polyA isolation kit we have used it the Illumina TruSeq RNA prep kit. You start with 4 ug of total RNA (not mRNA), follow the kit directions except:
(1) reduce the fragmentation time from 8 minutes to -- well, something less. Ignore the appendix in the back of the protocol, it is completely hosed -- no relationship between the sizes/times they give and what you actually get. If you are willing to mechanically fragment your cDNA you could skip the heat incubation altogether. Otherwise, seems like something around 30 seconds would be okay for a 454 run.
(2) Obviously, use 454 adapters, not Illumina adapters. You want the rapid adapters (with the "t" overhang), to fit with the Illumina protocol.
(3) For the 454 you use the unamplified library, so skip the enrichment PCR step.

The down side is that it typically takes Illumina a long time (months) to ship this kit after you order it. Also, per sample, it has a reasonable cost, but since it is meant to process 96 samples, it costs quite a bit.

--
Phillip

[maiyosry29]

Thanks pmiguel for your reply

However, we already have the Oligotex mRNA extraction kit in the lab and it give us a very low conc for mRNA which is not enough to have 200 ng/19 microlitre as the protocol states!!

So, any help reagrding this issue?

[maiyosry29]

Thanks pmiguel for your reply

However, we already have the Oligotex mRNA extraction kit in the lab and it give us a very low conc for mRNA which is not enough to have 200 ng/19 microlitre as the protocol states!!

So, any help reagrding this issue?

[mapplebe]

Is the problem just concentration? Do you have enough yield? You can do an EtOH precipitation (throw in some RNAse-free glycogen so you can get a firm visible pellet).

[Olaf Blue]

Remember that the mRNA component of the total RNA is only 1-5% depending on the cell type and organism. So if you start with 5-10 µg total RNA your mRNA yield (absent the small RNA after an oligo-dT enrichment) will be between aboyt 50 and 500 ng. That should be quite enough for RNA-Seq.

[pmiguel]

Is the problem just concentration? Do you have enough yield? You can do an EtOH precipitation (throw in some RNAse-free glycogen so you can get a firm visible pellet).

Better yet, avoid EtOH precipitations at all costs when your concentrations are low. Great way to lose most of your sample. Go with a Zymo column or even speed vap (no heat), if you must.

--
Phillip

[pmiguel]

Remember that the mRNA component of the total RNA is only 1-5% depending on the cell type and organism. So if you start with 5-10 µg total RNA your mRNA yield (absent the small RNA after an oligo-dT enrichment) will be between aboyt 50 and 500 ng. That should be quite enough for RNA-Seq.

I agree that it should be. Especially since you only need a few million molecules recast as amplicons. Sadly the Roche Rapid RNA method works poorly with less than 100-200 ng of RNA. Unclear why.

Not good for some samples -- because I have seen plenty of tissues where I wish I could get 1% yields of mRNA.

--
Phillip