mRNA on Bioanalyser

[dobbr493]

Hi there

We are looking at doing a transcriptome experiment with very limited starting material (usually around 10ug total RNA of good quality RIN > 8). We have tried both the Roche magnetic bead kit and Ambion spin column kit for extracting mRNA from total RNA and are unsure as to what the mRNA should look like on the Agilent Bioanalyser. An example of what we have been seeing is attached - is this mRNA or just rubbish?

Cheers

[RCJK]

I'd say that's just rubbish. What does the ladder lane look like? Did you run any blank lanes to see if the noisy baseline flattens out?

[RCJK]

Also, after those kits, are your samples in a buffer that is compatible with the Bioanalyser assay you are running?

[mnkyboy]

mRNA should look like an evenly distributed mound without the ribosomal peaks (I cant find an image easily). What you are showing is noise and either you concentration is too low (or there is nothing there) and/or your buffer is incompatible. You can see that type of result if your salt concentration is too high.

[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!

[pmiguel]

Yes that is just background noise. Your nano chip is not sensitive enough to detect your mRNA. You could run your mRNA undiluted on a RNA Pico chip that might pick something up. Or you could check fluorometrically (with a single stranded fluor) to get an idea of what your mRNA concentration is.

Maybe you have less total RNA to start with than you think? For the Nano chip you ran on the total RNA, what was the chip estimate of the concentration of your total RNA concentration? One common issue with estimating total RNA amounts is that even tiny amounts (1 ul per ml) of phenol will give you very strong readings at 260 nm, even in the absence of any nucleic acids.

--
Phillip

[shurjo]

I'm attaching a picture from poly(A)-selected mRNA (from 10ug of total RNA) run on a Pico Chip. As you can see, the ribosomal molecules are still noticeably present, but then those are very hard to remove completely.

[sisch]

mRNA should look like an evenly distributed mound without the ribosomal peaks (I cant find an image easily). What you are showing is noise and either you concentration is too low (or there is nothing there) and/or your buffer is incompatible. You can see that type of result if your salt concentration is too high.

Find attached one of our result files. To the left is totalRNA and to the right is purified mRNA with 1&2 being Qiagen Oligotex Kit purified (Pain in the ass imho) 3&4 Invitrogen Dynabeads.
So that's what we believe mRNA should look like on a Bioanalyzer (up to 8% of residual rRNA rendered no problem in our experiments)

By the way, we usually start off with 30 - 70ug totalRNA and have 0.5ug mRNA afterwards. The limit for sequencing w/o amplification is to my knowledge about 200ng mRNA.

Cheers,
Simon

[dobbr493]

I'm attaching a picture from poly(A)-selected mRNA (from 10ug of total RNA) run on a Pico Chip. As you can see, the ribosomal molecules are still noticeably present, but then those are very hard to remove completely.

That's very interesting - what was the kit that you used with that amount of total RNA? Also, what was the end-quantity of mRNA? Thanks for your help!

[shurjo]

That's very interesting - what was the kit that you used with that amount of total RNA? Also, what was the end-quantity of mRNA? Thanks for your help!

I used the DynaBeads kit from Invitrogen with 2 rounds of purification. mRNA yield from 10ug of total RNA with RIN=10 was about 120ng.

HTH,

Shurjo

[Ecap]

Hello,

I've observed bioanalyzer signatures very similar to yours when I've analyzed post-poly(A) selected total RNA samples in high salt and/or incompatible buffers.

For me, ethanol precipitation followed by sample reconstitution in a clean buffer (or water) totally solved the problem.

[Ecap]

Hello,

I've observed bioanalyzer signatures very similar to yours when I've analyzed post-poly(A) selected total RNA samples in high salt and/or incompatible buffers.

For me, ethanol precipitation followed by sample reconstitution in a clean buffer (or water) totally solved the problem.

I forgot to mention that in this case I was also using samples whose concentration were equal to or lesser than yours