RNA-Seq Sample Question

mlai2567 · 01-18-2019, 10:35 AM

Hi all,

I am preparing samples to send for RNA-sequencing. I've transformed a majority of my samples to cDNA, keeping only a few microliters of highly concentrated RNA for QC analysis, as I had concerns over the RNA stability. However, on the sequencing service page, it seems as if most samples are sent in RNA form. Has anyone successfully sent cDNA samples for RNA-sequencing?

Thanks in advance,
Michael

BioDynami · 01-18-2019, 12:58 PM

The commercial RNA-Seq kits are using RNA as input, not cDNA. You have converted RNA to cDNA, the single-stranded DNA? It may be a problem for the sequencing cores.

Bukowski · 01-19-2019, 08:31 AM

Of course you can submit cDNA for sequencing, at this point it's just 'DNA'. Most cores would probably prefer to take the RNA because then the library preparation is in their hands, and not yours - and they can do the QC that follows the process to maximise their confidence in the seqeuncing of your samples.

You just need to speak to the provider and tell them what the DNA source is.

Next time, just send the RNA.

luc · 01-19-2019, 06:26 PM

Bukowski is correct. RNA-seq library prep protocols (starting from RNA) are optimized for consistency to allow quantitative DGE analysis. Your cDNA prep protocol might not be.
Is your cDNA double-stranded (as required for most DNA library preps) or single-stranded?

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01-18-2019, 10:35 AM	#1
mlai2567 Junior Member Location: New York Join Date: Jan 2019 Posts: 1	RNA-Seq Sample Question Hi all, I am preparing samples to send for RNA-sequencing. I've transformed a majority of my samples to cDNA, keeping only a few microliters of highly concentrated RNA for QC analysis, as I had concerns over the RNA stability. However, on the sequencing service page, it seems as if most samples are sent in RNA form. Has anyone successfully sent cDNA samples for RNA-sequencing? Thanks in advance, Michael

01-18-2019, 12:58 PM	#2
BioDynami Registered Vendor Location: Alabama Join Date: Jul 2016 Posts: 17	The commercial RNA-Seq kits are using RNA as input, not cDNA. You have converted RNA to cDNA, the single-stranded DNA? It may be a problem for the sequencing cores.

01-19-2019, 08:31 AM	#3
Bukowski Senior Member Location: Aberdeen, Scotland Join Date: Jan 2010 Posts: 373	Of course you can submit cDNA for sequencing, at this point it's just 'DNA'. Most cores would probably prefer to take the RNA because then the library preparation is in their hands, and not yours - and they can do the QC that follows the process to maximise their confidence in the seqeuncing of your samples. You just need to speak to the provider and tell them what the DNA source is. Next time, just send the RNA.

01-19-2019, 06:26 PM	#4
luc Senior Member Location: US Join Date: Dec 2010 Posts: 360	Bukowski is correct. RNA-seq library prep protocols (starting from RNA) are optimized for consistency to allow quantitative DGE analysis. Your cDNA prep protocol might not be. Is your cDNA double-stranded (as required for most DNA library preps) or single-stranded?