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Old 10-10-2012, 09:32 AM   #1
cah
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Location: Minnesota, US

Join Date: Aug 2012
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Default FFPE RNA library prep and capture

We are looking for a way to improve the data we obtain from FFPE RNA samples that we are sequencing (Illumina).

Knowing the FFPE samples are degraded, we assume poly A tails are not necessarily present on the mRNA for these samples which is effectively reducing the coverage we see. We are looking for an effective work around.

I've read some positive results about Ribozero and DSN normalization, but am told that these methods have been tested here without great results.

Since we are working with FFPE samples, we were thinking that we would not do any fragmentation or poly A selection and simply start with cDNA synthesis, make ds cDNA and then capture what we are interested in. The problem is we are interested in as much of the whole transcriptome that is there. We are looking at using an Agilent SureSelect kit, but they don't make a Whole Transcriptome RNA capture kit. They do make a kinome kit and custom kits, but custom could be expensive and time consuming.

In theory, why wouldn't the Agilent SureSelect All Exon (DNA) capture kit work on a ds cDNA library? It seems like the baits that are specific to the Exome, should be complimentary to the specific strands of mRNA that we made cDNA out of and leave the rest behind.

Maybe I'm missing something obvious though.
Has anyone tried this?
Does anyone have any recommendations/hints/advice for doing this?

Last edited by cah; 10-10-2012 at 12:50 PM.
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Old 02-14-2013, 04:57 AM   #2
veronica
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Location: Italy

Join Date: Sep 2010
Posts: 3
Default

Quote:
Originally Posted by cah View Post
We are looking for a way to improve the data we obtain from FFPE RNA samples that we are sequencing (Illumina).

Knowing the FFPE samples are degraded, we assume poly A tails are not necessarily present on the mRNA for these samples which is effectively reducing the coverage we see. We are looking for an effective work around.

I've read some positive results about Ribozero and DSN normalization, but am told that these methods have been tested here without great results.

Since we are working with FFPE samples, we were thinking that we would not do any fragmentation or poly A selection and simply start with cDNA synthesis, make ds cDNA and then capture what we are interested in. The problem is we are interested in as much of the whole transcriptome that is there. We are looking at using an Agilent SureSelect kit, but they don't make a Whole Transcriptome RNA capture kit. They do make a kinome kit and custom kits, but custom could be expensive and time consuming.

In theory, why wouldn't the Agilent SureSelect All Exon (DNA) capture kit work on a ds cDNA library? It seems like the baits that are specific to the Exome, should be complimentary to the specific strands of mRNA that we made cDNA out of and leave the rest behind.

Maybe I'm missing something obvious though.
Has anyone tried this?
Does anyone have any recommendations/hints/advice for doing this?
For what I have heard Illumina's R&D is doing something like that.. but it is second hand news...
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Old 06-23-2015, 04:54 PM   #3
Him26
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Location: California US

Join Date: Aug 2011
Posts: 18
Default

Quote:
Originally Posted by cah View Post
We are looking for a way to improve the data we obtain from FFPE RNA samples that we are sequencing (Illumina).

Knowing the FFPE samples are degraded, we assume poly A tails are not necessarily present on the mRNA for these samples which is effectively reducing the coverage we see. We are looking for an effective work around.

I've read some positive results about Ribozero and DSN normalization, but am told that these methods have been tested here without great results.

Since we are working with FFPE samples, we were thinking that we would not do any fragmentation or poly A selection and simply start with cDNA synthesis, make ds cDNA and then capture what we are interested in. The problem is we are interested in as much of the whole transcriptome that is there. We are looking at using an Agilent SureSelect kit, but they don't make a Whole Transcriptome RNA capture kit. They do make a kinome kit and custom kits, but custom could be expensive and time consuming.

In theory, why wouldn't the Agilent SureSelect All Exon (DNA) capture kit work on a ds cDNA library? It seems like the baits that are specific to the Exome, should be complimentary to the specific strands of mRNA that we made cDNA out of and leave the rest behind.

Maybe I'm missing something obvious though.
Has anyone tried this?
Does anyone have any recommendations/hints/advice for doing this?
Hi, I am interested in performing RNAseq on FFPE samples. I see lots of papers on google but I was wondering if you would share your experience. Thank you.
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