Can I use degraded total-RNA for mRNA-Seq library production?
The use of degraded RNA will have a large effect on the performance of the assay. One of the first steps in the process is the purification of poly-A mRNA using a poly-T capture step. If the RNA is degraded, the mRNA that is captured at this step will not be full length, and thus will not be able to give full-length cDNA products after random priming. When we use high-quality total RNA, we are able to produce even, end-to-end coverage of each mRNA molecule present in the sample. However, if the RNA is degraded, you will probably observe a noticeable 3′- to 5′-bias in the number of reads observed for most transcripts. If you do not have a choice, and the best you can do from your system is degraded RNA, you can certainly still use this process to create libraries for sequencing on the Genome Analyzer. The more the RNA is degraded, the stronger the 3′-bias you will see in the sequencing. For this reason, when trying to compare expression levels across many samples, it is important to com
