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Old 05-29-2015, 12:30 PM   #1
PhatB
Junior Member
 
Location: San Diego

Join Date: May 2015
Posts: 3
Default 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!
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