Problems with Globin genes in mRNAseq paired-end library preparation

[div1982]

Hello everyone
Was wondering if anybody had problems with excess of globin genes fragments during an mRNAseq library preparation?
More than 60% of fragments (picked at random for quality testing) turned out to be alpha and beta globin genes. Is this normal?
This is of course whole blood RNA so I am wondering if it makes sense to do globin reduction?
If anyone has any experiences, it would be greatly appreciated.
Kind regards
D

[Melissa]

Hello everyone
Was wondering if anybody had problems with excess of globin genes fragments during an mRNAseq library preparation?
More than 60% of fragments (picked at random for quality testing) turned out to be alpha and beta globin genes. Is this normal?
This is of course whole blood RNA so I am wondering if it makes sense to do globin reduction?
If anyone has any experiences, it would be greatly appreciated.
Kind regards
D

Try cDNA normalization.

[Melissa]

http://www.evrogen.com/technologies/normalization.shtml

[elaney_k]

Have you tried using a beta-globin reduction protocol?
The evrogen kit that Melissa provided the link for reduces the most abundantly expressed transcripts in a fairly random fashion. There are beta-globin reduction protocols that have been used for samples that were undergoing microarray analysis to specifically target and reduce the amount of beta-globin mRNA transcripts. The principles used for the two methods are pretty similar. Ambion have a kit called GLOBINclear and Affymetrix have a protocol http://www.affymetrix.com/support/te...2_technote.pdf, you'll probably find other kits/methods online also.

Elaine

[div1982]

Thanks for the replies, yes I am aware of the Globinclear kit and we have used it for gene expression. However, my concern here is the effect of the globin in NGS.For microarray-based gene expression it is known that globin is a major problem. I have not read anywhere that people have combined globin reduction with NGS workflow as yet.
I will soon have some results that might shed some light on this, will keep you posted
Cheers