Whole Blood RNA-Seq Libraries and globins

[jbrowne73]

I am planning to prepare some RNA-Seq libraries using RNA derived from whole blood (Bos taurus) extracted using the Tempus system from Applied Biosystems. I am concerned that the libraries will contain a large number of transcripts derived from globin mRNA. I have two questions
1 Does anyone have an estimate of the percentage of transcripts that will align to globin?
2 Does anyone have any opinions/advice on the best way to remove the globins either using a globin specific kit or a DNS method.

All advice welcomed.

[whw]

This paper may help: http://www.springerlink.com/content/c44u853010k2p610/

I haven't tried this approach myself but I would be interested to hear if it works.

[kfitch]

In humans, about 70% of mRNA from blood is hemoglobin. My colleagues and I have tried DSN and successfully removed hemoglobin and ribosomal transcripts from total blood RNA, however other abundant transcripts are also affected by the treatment.
There is a service provider http://www.prognosysbio.com that will do the DSN treatment for you, and their website has some useful info on the protocol. We have not used the service.

[Veleno]

How about RBC lysis before extracting your RNA?

[lmolokin]

GLOBINclear

I have used the GLOBINclear kit (Ambion/Life Tech) to reduce globin mRNA (HBA1/HBB) in total RNA from human blood. In my experience, most of the time the reduction works and you see less than 5% globin content after mapping. Other times the globin content can remain as high as 50% of total gene "counts". No matter how careful the reduction protocol is carried out it's still a mystery to me as to why it produces inconsistent results.

[masterpiece]

I found this recently published paper discussion on effect on transcripts detection after globin removal. Might be useful to you guys.

http://www.biomedcentral.com/content/pdf/1471-2164-13-28.pdf

They use GLOBINclear kit (Ambion/Life Tech) for globin removal.

[Joann]

Thank you for pointing out this paper.

[aniruddha.otago]

Hey Guys,

I am aiming to perform RNA-seq from human Neutrophils. I am taking peripheral blood and the enriching for Neutrophils ( Ficoll+ NH4cl and also tried Ficoll+dextran and even tried Ficoll+hypotonic treatment). In all cases I am getting good enrichment (measured in Heamatology cell analyser, CBC count). Then I went on to isolate the total RNA from the neutrophil enriched pellet. Trizol+Ambion kit. Checked in bioanalyser they are degraged. I am not getting whats going wrong. 10 ml blood, so there are heaps of cells. Any idea, advice please.

[Élodie]

I also use GLOBINclear from Ambion and I have no problem with this kit. I work on human whole blood in Tempus tube and do total RNA extraction with ABI kit or the ABI MagMax. Then, I deplete globin mRNA with the GLOBINclear kit. And finally, I use TruSeq RNA sample preparation kit from Illumina. The GLOBINclear kit uses oligos which hybridize to Globin mRNA, and streptavidin magnetic beads which hybridize to oligos. So you can remove it with a magnetic stand.

[jbrowne73]

Globin Contamination

Just to follow up on my first question, we went ahead with library preparation without any globin removal and the results are excellent with tiny amounts of globin contamination(>0.5%). It seems that globin contamination in Bos taurus is not the same problems as in humans due to the very small numbers of reticulocytes present in healthy cows compared to some other mammals.

[Veleno]

Hey Guys,

I am aiming to perform RNA-seq from human Neutrophils. I am taking peripheral blood and the enriching for Neutrophils ( Ficoll+ NH4cl and also tried Ficoll+dextran and even tried Ficoll+hypotonic treatment). In all cases I am getting good enrichment (measured in Heamatology cell analyser, CBC count). Then I went on to isolate the total RNA from the neutrophil enriched pellet. Trizol+Ambion kit. Checked in bioanalyser they are degraged. I am not getting whats going wrong. 10 ml blood, so there are heaps of cells. Any idea, advice please.

If you are after reference RNA seq dataset the Blueprint consortium released at least 6 samples, plus 200 will follow.

If you are interested in the protocol I think they have it online on their website and apparently they do trizol and then precipitate it with RIN>8.5

[NuGEN]

We have recently launched a stranded RNA-Seq kit developed specifically for working with human whole blood total RNA. The workflow minimizes globin and rRNA sequencing reads by targeted depletion of the adaptor ligated library. We have data available from Human PAXgene samples here - http://www.nugeninc.com/nugen/index....ore/encore-wb/