RNA-Seq Library Prep from PAXgene RNA Blood Tubes

gmassacc · 04-16-2020, 02:36 PM

Hi all,

My group is starting an RNA-seq project from patient frozen blood stored in PAXgene RNA blood collection tubes. We have experience performing library preps on PBMCs and isolated monocytes using the Takara Bio SMARTer RNA-seq kits. For this project, we have heard that one issue that could arise is the globin gene counts after sequencing. Has anybody had any issues with this?

Right now, I am thinking about extracting RNA from the PAXgene tube using the QIAGEN PAXgene extraction kit, then using this kit: GLOBINclear Kit for globin mRNA depletion and then running the standard library prep kits we have in the past.

However, Illumina sells a library prep kit that removes globin mRNA in the library prep itself: https://www.illumina.com/products/by...na-globin.html

Does anybody have any experience with this kit?

Thanks for all your help!

luc · 04-16-2020, 04:20 PM

A secret tip:
https://www.qiagen.com/us/products/d...inginformation
Much easier than any other solution.

nucacidhunter · 04-17-2020, 01:23 AM

Illumina kit is gold standard in this application if RNA quantity is not limiting. On the other hand performance of Qiagen kit varies and I would suggest to trial it before committing to use it.

jteeee2 · 04-17-2020, 06:29 AM

Agree with @nucacidhunter. We were initially excited about the new Qiagen product but have also seen some odd performance issues...particularly when including globin-reduction probes. The Illumina TruSeq kit is solid. I've had good success with the NEBNext Ultra II Directional RNA kits with globin depletion. NEB also has a protocol for globin depletion followed by poly-A enrichment if you were interested in limiting transcript coverage to mRNAs.

gmassacc · 04-17-2020, 07:36 AM

Thanks for the replies! I will look into the NEB kit and the Illumina!

jteeee2 · 04-17-2020, 10:38 AM

There's also this paper that came out a few days ago which suggests you don't need to deplete globin at all if you're starting with Paxgene blood.
https://www.nature.com/articles/s41598-020-62801-6

I'd probably err on the side of caution and go ahead with your depletion strategy

It won't hurt.

gmassacc · 04-20-2020, 07:04 AM

Super informative paper! Thanks jteeee2!

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04-16-2020, 02:36 PM	#1
gmassacc Junior Member Location: Baltimore, MD Join Date: Apr 2020 Posts: 3	RNA-Seq Library Prep from PAXgene RNA Blood Tubes Hi all, My group is starting an RNA-seq project from patient frozen blood stored in PAXgene RNA blood collection tubes. We have experience performing library preps on PBMCs and isolated monocytes using the Takara Bio SMARTer RNA-seq kits. For this project, we have heard that one issue that could arise is the globin gene counts after sequencing. Has anybody had any issues with this? Right now, I am thinking about extracting RNA from the PAXgene tube using the QIAGEN PAXgene extraction kit, then using this kit: GLOBINclear Kit for globin mRNA depletion and then running the standard library prep kits we have in the past. However, Illumina sells a library prep kit that removes globin mRNA in the library prep itself: https://www.illumina.com/products/by...na-globin.html Does anybody have any experience with this kit? Thanks for all your help!

04-16-2020, 04:20 PM	#2
luc Senior Member Location: US Join Date: Dec 2010 Posts: 452	A secret tip: https://www.qiagen.com/us/products/d...inginformation Much easier than any other solution.

04-17-2020, 01:23 AM	#3
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	Illumina kit is gold standard in this application if RNA quantity is not limiting. On the other hand performance of Qiagen kit varies and I would suggest to trial it before committing to use it.

04-17-2020, 06:29 AM	#4
jteeee2 Member Location: Illinois Join Date: Oct 2014 Posts: 42	Agree with @nucacidhunter. We were initially excited about the new Qiagen product but have also seen some odd performance issues...particularly when including globin-reduction probes. The Illumina TruSeq kit is solid. I've had good success with the NEBNext Ultra II Directional RNA kits with globin depletion. NEB also has a protocol for globin depletion followed by poly-A enrichment if you were interested in limiting transcript coverage to mRNAs.

04-17-2020, 07:36 AM	#5
gmassacc Junior Member Location: Baltimore, MD Join Date: Apr 2020 Posts: 3	Thanks for the replies! I will look into the NEB kit and the Illumina!

04-17-2020, 10:38 AM	#6
jteeee2 Member Location: Illinois Join Date: Oct 2014 Posts: 42	There's also this paper that came out a few days ago which suggests you don't need to deplete globin at all if you're starting with Paxgene blood. https://www.nature.com/articles/s41598-020-62801-6 I'd probably err on the side of caution and go ahead with your depletion strategy It won't hurt.

04-20-2020, 07:04 AM	#7
gmassacc Junior Member Location: Baltimore, MD Join Date: Apr 2020 Posts: 3	Super informative paper! Thanks jteeee2!