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Old 06-08-2018, 09:59 AM   #259
Location: Bay Area

Join Date: Jun 2012
Posts: 95

Originally Posted by ChristmasSunflower View Post

I recently used Smart_Seq2 protocol for single cell study. These single cell is manually picked up into 8-strip PCR tube. I designed all 3 primers TSO, Oligo-dT30VN, and ISPCR oligo with biotin modification in 5' side. I did a few of experimental samples, along with RNA positive control RNA 1ng, 100pg and 10pg, and water negative control. I did 18 PCR cycles for preamplification and 1:1 beads ratio for PCR cleanup. From Qubit reading, it's hard to say if protocol worked. I run them with Bioanalyzer. It seems results are positive. Can someone here with more experimental experience comment this result?

There's tons of low MW products there indicating massive RNA degradation, which you have in both your cell and purified RNA samples. Did you check the RIN of your pure RNA? If it's high, then you've got an RNase contamination problem, otherwise you need to look at your cell/RNA isolation procedure. I would not proceed to library prep and sequencing with those samples.
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