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Old 06-22-2011, 09:31 PM   #1
dobbr493
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Default mRNA on Bioanalyser

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
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File Type: pdf 2100 expert_mRNA Nano_DE04103720_2011-06-20_12-31-53.pdf (859.3 KB, 179 views)
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Old 06-22-2011, 11:17 PM   #2
RCJK
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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?
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Old 06-22-2011, 11:18 PM   #3
RCJK
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Also, after those kits, are your samples in a buffer that is compatible with the Bioanalyser assay you are running?
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Old 06-23-2011, 09:50 AM   #4
mnkyboy
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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.
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Old 07-07-2011, 05:13 PM   #5
dobbr493
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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!
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Old 07-08-2011, 08:12 AM   #6
pmiguel
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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.

--
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Old 07-08-2011, 08:15 AM   #7
shurjo
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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.
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Old 07-08-2011, 08:51 AM   #8
sisch
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Quote:
Originally Posted by mnkyboy View Post
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
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Last edited by sisch; 07-08-2011 at 08:53 AM.
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Old 07-10-2011, 05:19 PM   #9
dobbr493
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Quote:
Originally Posted by shurjo View Post
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!
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Old 07-11-2011, 09:03 AM   #10
shurjo
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Quote:
Originally Posted by dobbr493 View Post
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
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Old 04-25-2012, 08:09 AM   #11
Ecap
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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.
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Old 04-25-2012, 08:09 AM   #12
Ecap
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Quote:
Originally Posted by Ecap View Post
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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