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-   -   RNA from FACS-sorted Para-fixed cells for RNA-seq (http://seqanswers.com/forums/showthread.php?t=59059)

PhatB 05-29-2015 12:30 PM

RNA from FACS-sorted Para-fixed cells for RNA-seq
 
Hey all,

I have recently attempted to FACS sort virally infected mouse splenocytes that were fixed and stained with intracellular flow antibodies. I yielded about 1.5 million cells of each population and subsequently utilized the Recoverall FFPE kit from Life Tech to isolate the RNA. This method has been recently described here: http://www.ncbi.nlm.nih.gov/pubmed/24594682 The authors of this study demonstrated RNA RIN scores above 8!

My ultimate goal is to utilize Illumina deep sequencing to compare gene expression analysis between infected vs. non-infected cell subsets. However, my total RNA yield was about 700-1000ng @ 15ng/ul and my RIN scores ranged from 2.8 to 3.8. Based on these results, is it possible to get reliable gene expression analysis via illumina with such low RIN and total RNA yields and if it still can work, what methodology would be recommended?

Thanks!

cmbetts 05-29-2015 01:09 PM

You have plenty of RNA, so your main issue is the low RIN scores. The key issue with low RIN RNA is that using common PolyA purification methods to isolate mRNA will lead to extreme 3' bias and other issues in your libraries. However, if you use an rRNA depletion method (Lots kits using either probe pull-down or RNaseH digestion are available) and skip any fragmentation steps in your library protocol, you should be fine.
I'd recommend checking out the literature for RNA-Seq from FFPE RNA and use their methods for guidance.

kmcarr 05-29-2015 03:18 PM

Quote:

Originally Posted by cmbetts (Post 173619)
You have plenty of RNA, so your main issue is the low RIN scores. The key issue with low RIN RNA is that using common PolyA purification methods to isolate mRNA will lead to extreme 3' bias and other issues in your libraries. However, if you use an rRNA depletion method (Lots kits using either probe pull-down or RNaseH digestion are available) and skip any fragmentation steps in your library protocol, you should be fine.
I'd recommend checking out the literature for RNA-Seq from FFPE RNA and use their methods for guidance.

Our lab recently used the NuGen Ovation RNA-Seq System for Model Organisms for some degraded mouse RNA sample (not FFPE, just lower quality) and it worked very well at eliminating rRNA. Unfortumately I don't have a comparable library prepared using a standard kit to assess any biases in mRNA representation.


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