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!
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!
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