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Old 02-19-2013, 09:46 PM   #1
Pengfei Liu
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Location: Beijing

Join Date: Nov 2012
Posts: 16
Question RNA quality of my samples, what to do next?

Recently I am working on the transcriptome of Methanocella conradii HZ254 (a methanogenic archaea isolated in our lab)
I have three culture systems:
1), HZ254 pure culture,
2), P. thermopropionicum pure culture with pyruvate as substrate and
3) coculture of HZ254& P. thermopropionicum with propionate as substrate.

I use Qiagen Allprep DNA/RNA mini kit for the isolation of RNA,
I got good RNA results of HZ254 pure culture (please see the attachment fig 1.) due to the easy disruption of the cell of HZ254, I just homogenized for 20s at speed 4.0m/s on fast prep without the addition of glass beads.
Same method failed to extract RNA of P. thermopropionicum, so I try following protocol, homogenized for 120 s (3*40 s. with 30 s intervals) in a FastPrep FP120 (Qbiogene) at speed 5.0 with the addition of glass beads. Compared with the result (please see the attachment fig 2) of the attached paper (genome of P. thermopropionicum @ genome research), I think it's also good, although the RIN was low.

So the coculture was followd the same lyse protocol, 120 s (3*40 s. with 30 s intervals) in a FastPrep FP120 (Qbiogene) at speed 5.0 with the addition of glass beads. the result seemed not good (please see the attachment fig 3&4.) I want to use these samples to do RNA-seq, the technian of our local sequencing center told me first, the Ratio of 23s/16s was lower than 1, and second the RIN was a little bit lower (which around 7).

So now I need to make sure whether the special features of the RNA of P. thermopropionicum (Fragmentation of 23S rRNA into three parts, as indicated in the attached paper) caused the lower 23s/16s ratio and lower RIN or the longer lyse caused the degradation of the coculture RNA sample.
(the RNA of P. thermopropionicum account for ~30% of the total RNA of the coculture, as indicated in another paper)


Please share your opinion on my results! RIN of HZ254 and coculture.pdf

Kosaka et al-2008-The genome of Pelotomaculum thermopropionicum-Genome Research.pdf

Kosaka et al-2008-The genome of Pelotomaculum thermopropionicum-Genome Research-SOM.pdf
Thank you very much!

Last edited by Pengfei Liu; 02-19-2013 at 09:48 PM.
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