Field of Science

So long since I've posted...

But here's an update on the RNA-seq saga.

We've been struggling to get evidence that our RNA preps are of good enough quality for library preparation and sequencing.  Because the Ribo-zero kits that we use to remove the rRNA from the samples are so expensive, we wanted to be sure the final mRNA concentrations and integrity would be good enough to make the equally expensive sequencing worthwhile.

Unfortunately my attempts at using a Bioanalyzer have been very frustrating (and it's not cheap - several $ per sample), with odd smeary/spiky patterns and poor recognition of the size standards. This is partly because the concentrations are so low and the 'pico-RNA kits are very fussy, and partly because it's just a fussy procedure and easy (for me) to make mistakes.

We sought advice from the person who will be preparing the sequencing libraries for us.  She said that samples that appeared bad on Bioanalyzer results often gave excellent sequencing results, and suggested that we have her prep an initial 8 libraries.  She would then pass these on to the person who does the sequencing, who could just run her initial quality checks on the libraries and tell us whether there were suitable for sequencing.  So that's what we did, and the news was good - these initial libraries look OK.

Now it's time to do the Ribo-zero and final clean-up on the rest of the samples, so they can be handed over for eventual library prep and sequencing.  There's a two-month backlog so we likely will get the results as a Christmas present.

Our sample processing had been a bit scattershot, so now I needed to go through the sample information and make sure we have the right samples, and that the numbers add up.  They do, so I think we're all set to quickly complete the remaining Ribo-zero treatments and use RNeasy-Min-elute columns to clean up each RNA sample after Ribo-zero.  Then the remaining 64 samples can be handed over.  (The 'we' here is our excellent UBC-Co-op program technician.)

We have accidentally wasted two of our Ribo-zero treatments on duplicate samples, and I was thinking that this meant that we'd have to buy another 6-sample kit ($650).  But now I think we can get by without this, either by not treating two samples (the G6 samples in the table above) or by using slightly less of the reagent for a subset of samples.

We had been using only 1 µg of total RNA for Ribo-zero treatment, but for these samples we'll sue all the DNase-treated RNA we have (typically several µg).  In some cases the volume of sample will be too high (max 28 µl for Ribo-zero), but singe the samples are in H2O we can just evaporate them a bit to decrease the volume.


  1. I had a similar experience with a bioanalyzer a couple years ago. I finally figured out that it's super sensitive to concentration and I was over-concentrating the RNA for the pico chip. I don't know if that's your problem, but I would make sure to use the proper concentration of RNA. If the concentration that the bioanalyzer reports is out of the range of the Pico chip, don't trust any result the bioanalyzer gives you.

  2. Hi Rosie, what concentration of RNA are you getting? The Pico chips are a bigger pain than the nano ones, but they should be working reliably - speak to Agilent if the ladder is not behaving!

    Are there options for using RNaseH degradation of rRNA rather than RibZero pull-out? We're looking at that here for Eukaryotes.


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