OHSU

Sample Preparation

The MPSSR can process genomic DNA and total RNA. There is no need to prepare polyA(+) RNA since the polyA(+) extraction is the first step in our protocol.

Standard protocols for nucleic acid extraction work well for producing material that can be used to construct a sequencing library. These protocols include, but are not limited to: Trizol, phenol-chloroform, and Qiagen spin columns. We have been receiving feedback that some of the newer all-prep kits tend to give lower quality RNA than focused kits, so please keep that in mind when selecting an isolation protocol.

Note, also, that some protocols do not work well for isolating microRNAs from a sample. If microRNA analysis is to be run, it is important to use a protocol that has been evaluated for retention of lower molecular weight RNAs.

All RNAs submitted to the MPSSR are evaluated for quality using a Bioanalyzer. We rely on a visual inspection of the Bioanalyzer trace combined with the RIN score to determine the quality of the material. RIN scores above 8 generally produce high quality libraries. We have been able to successfully make libraries from samples with RIN scores between 6 and 8, but samples with RIN scores below 6 do not reliably produce quality data.