polyA selection kits

[chaos81]

Can someone please point me in the direction of an RNA polyA selection kit that has an input of less than 2ug. We are currently using the Illumina method, but would like to compare to another platform. At this point the lowest input I can locate is 2ug and typically work with samples around 1-2ug of total RNA. Thanks.

[protist]

We use Dynabeads from Invitrogen - either the mRNA kit (Beads plus buffers) or the Dynabeads Oligo(dT)25 with homemade buffers. Starting amounts of RNA are generally 1-3 ug of total RNA.

[chaos81]

Thanks Protist. Also, when you use the mRNA kit do you change anything up regarding the protocol or go with what they suggest?

[protist]

We do two rounds of polyA - see attached PDF for detail

[chaos81]

Great! do you have an idea of what percent yield you typically see per 1ug? just curious but thanks for the help. We are going to try this method soon.

[protist]

Dependent on the organism in our hands it would be about 2-8% (~20-80 ng of polyA from 1 ug total). I don't tend to quantify my polyA as generally there are no problems and when I start with very small amounts I would rather feed it all to fragmentation & cDNA synthesis reactions - for new RNAs we do quantify the cDNA generated.

[jmRNA]

Hi Protist, thanks for posting your protocol, I've been using a similar one, but am going to try yours as I'm getting inconsistent results.

What levels of rRNA contamination were you seeing after polyA selection with the Dynabeads? I've seen anywhere from 0-7%, but I've generally only proceeded into library prep if I have less than 2%. What is your threshold?

thanks

[protist]

Originally Posted by jmRNA

What levels of rRNA contamination were you seeing after polyA selection with the Dynabeads? I've seen anywhere from 0-7%, but I've generally only proceeded into library prep if I have less than 2%. What is your threshold?

For the polyA selected material we see 0- 2% - it just seems to work very efficiently (samples are unicellular eukaryotes, human, viral and yeast). We also do a lot of bacterial work but use rRNA depletion for that as their mRNAs don't have nice convenient polyA tails!

We have been doing RNAseq since 2008 we have not substantially changed the polyA protocol or switched the beads - so we don't really have a threshold as we have yet to see sizable rRNA contamination,

As a comparison on the threshold level, even after rRNA depletion treatment for some our bacterial samples we have had to deal with sometimes >80% rRNA contamination. For the bacterial samples we changed to to RiboZero in the last year and I cannot recommend it highly enough. With the other rRNA kits the typical 50-80% rRNA contamination that carried through is now reduced to ~2-10% depending on the Beastie in question.

One trick is to be sure you leave the beads on the magnet for a decent amount of time at lest 2-3min. If you see any tinge of brown in your mRNA put it back on the magnet. A couple of times I noticed bead carryover in my fragmented RNA and have put it back on a magnet before proceeding.

Best of luck I hope that the selection works well.

[jmRNA]

Thanks for the confirmation on the %rRNA contamination. I glad to hear that RiboZero works well, I will be testing it out soon, however our samples are human.