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Using degraded RNA with TotalRNA Seq Kit
The Total RNA Seq Kit (Ambion/LifeTech) is designed for constructing SOLiD RNA seq libraries. It produces strand-specific libraries by ligating the 5'-end of RNA fragments to 5'-end adapters[1]. This ligation requires the 5'-end of the RNA to be mono-phosphorylated. The ligation works because the RNAse chosen by Ambion to fragment the RNA prior to ligation is of a type that leaves a 5'-phosphate upon cleavage.
This idea occurred to me recently--I am speculating, not reporting experimental results.
One possible issue is that some RNA degradation may have occurred prior to the RNA fragmentation step. However, it is my understanding that most RNAses and chemical/heat modes of RNA degradation do not leave a 5'-monophosphate end. Rather they proceed via nucleophilic attack of the 2'-OH group on the phosphate backbone and result in 5'-hydroxyl, 3'-phosphate. Right?
If so, these fragments will not be substrates for ligation to the adapters provided in the Total RNA Seq kit.
This is not bad, per se, just something to keep in mind. If you did want to make an RNA Seq library from fairly heavily degraded RNA, you would want to treat it with an enzyme such as T4-polynucleotide kinase (PNK) prior to ligation. Subsequent removal of PNK might also be critical because it could otherwise allow increased amounts of adapter-dimers to form. (Or not. I do not know the details of the structure of these adapters.)
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Phillip
[1]Note that this implies that to ligate to the 5' ends of capped RNA molecules, those RNAs would need to be de-capped (with, eg, Tabacco Acid Pyrophosphatase) first.
This idea occurred to me recently--I am speculating, not reporting experimental results.
One possible issue is that some RNA degradation may have occurred prior to the RNA fragmentation step. However, it is my understanding that most RNAses and chemical/heat modes of RNA degradation do not leave a 5'-monophosphate end. Rather they proceed via nucleophilic attack of the 2'-OH group on the phosphate backbone and result in 5'-hydroxyl, 3'-phosphate. Right?
If so, these fragments will not be substrates for ligation to the adapters provided in the Total RNA Seq kit.
This is not bad, per se, just something to keep in mind. If you did want to make an RNA Seq library from fairly heavily degraded RNA, you would want to treat it with an enzyme such as T4-polynucleotide kinase (PNK) prior to ligation. Subsequent removal of PNK might also be critical because it could otherwise allow increased amounts of adapter-dimers to form. (Or not. I do not know the details of the structure of these adapters.)
--
Phillip
[1]Note that this implies that to ligate to the 5' ends of capped RNA molecules, those RNAs would need to be de-capped (with, eg, Tabacco Acid Pyrophosphatase) first.