Carrier RNA and Illumina sequencing

[kga1978]

Hi All,

We have recently experienced a lot of problems with our RNA library preps when using samples containing a large amount of Qiagen carrier RNA (poly-A, 100bp-10kb in length). Specifically, we see that a very strong band migrating at 4kb can be observed after the initial steps of RT (using random hexamers) and second-strand synthesis. We now know that this band is created by the presence of carrier RNA and we have been unable to properly fragment it using the standard RNA fragmentation protocols. The presence of this band is problematic as we have found that it is necessary to perform several additional cycles of enrichment PCR in order to get libraries of a reasonable size.

The question is - have any of you experienced problems when using samples containing carrier RNA? Have you found solutions to the problem? We would rather not have to use (early stage) size selection and pulling out the carrier RNA using something like poly-dT isn't a viable option due to off-target effects (the carrier RNA concentration is two-three orders of magnitude more than what we are trying to sequence). We have also thought about using RNAse H-based methods or modified random hexamers without 'T' in the last two positions, but other than that we are out of ideas.

Any thoughts?

Thanks very much in advance!

[owenman]

This is an old question but a colleague and I recently posed a similar question to Illumina tech support and I thought I would post their reply:

Q: Do we know if the use of carrier RNA during RNA extraction (such as MS2 bacteriophage RNA), to increase RNA yield, is incompatible with any Illumina library prep/sequencing methods? I want to know if I should abandon the use of carrier RNA.

A: We have not tested in house if carriers like glycogen or LPA or MS2 bacteriophage RNA would interfere with our sample prep . It appears from some customers' experience that when using nucleic acid carriers, a large chunk (50-90%) of the data output was made up by the carrier molecule sequence.

Our Truseq RNA sample prep purifies the poly-A containing mRNA molecules using poly-T oligoattached magnetic beads prior to fragmentation and adaptor ligation etc. You may like to check if the MS2 bacteriophage RNA has poly A sequences, and if so, your carrier RNA might generate libraries as the carrier RNA will also enter the workflow along with the purified mRNA; but depending on the size they might be smaller and fragmented to much smaller sizes then your library of interest.

In essence, we cannot officially recommend that you use the carrier RNA as we do not know how it will affect the outcome. Nonetheless, if you wish to try, I would personally suggest you try out with 1-2 samples to see how it works. It may also be better to use non nucleic acid carriers.