mRNA on Bioanalyser

dobbr493 · 06-22-2011, 09:31 PM

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 · 06-22-2011, 11:17 PM

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 · 06-22-2011, 11:18 PM

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

mnkyboy · 06-23-2011, 09:50 AM

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 · 07-07-2011, 05:13 PM

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 · 07-08-2011, 08:12 AM

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 · 07-08-2011, 08:15 AM

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 · 07-08-2011, 08:51 AM

Quote:

Originally Posted by 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.

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 · 07-10-2011, 05:19 PM

Quote:

Originally Posted by 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.

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 · 07-11-2011, 09:03 AM

Quote:

Originally Posted by dobbr493

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 · 04-25-2012, 08:09 AM

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 · 04-25-2012, 08:09 AM

Quote:

Originally Posted by 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

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06-22-2011, 11:17 PM	#2
RCJK Senior Member Location: Australia Join Date: May 2009 Posts: 155	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?

06-22-2011, 11:18 PM	#3
RCJK Senior Member Location: Australia Join Date: May 2009 Posts: 155	Also, after those kits, are your samples in a buffer that is compatible with the Bioanalyser assay you are running?

06-23-2011, 09:50 AM	#4
mnkyboy Member Location: Seattle, WA Join Date: Mar 2009 Posts: 87	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.

07-07-2011, 05:13 PM	#5
dobbr493 Junior Member Location: New Zealand Join Date: Jun 2011 Posts: 5	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!

07-08-2011, 08:12 AM	#6
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	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

04-25-2012, 08:09 AM	#11
Ecap Junior Member Location: Maryland Join Date: Apr 2012 Posts: 2	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.