RNA isolation for small RNA Next-Generation Sequencing from acellular biofluids.

There are a number of considerations when choosing protocols both upstream and downstream of Next-Generation Sequencing experiments. On the front end, purification methods, additives, and residuum can often inhibit the sensitive chemistries by which sequencing-by-synthesis is performed. On the back end, data handling, analysis software packages, and pipelines can also impact sequencing outcomes. The current chapter will describe stepwise how acellular biofluid samples are prepared for small RNA sequencing. With regard to purification methods, we found that small RNA yield can be improved considerably by following the total RNA isolation protocol included with Ambion's mirVana PARIS Kit but modifying the organic extraction step. Specifically, after transferring the upper aqueous phase to a fresh tube, water is added to the residual material (interphase and lower organic layer) and again phase-separated. In contrast, all the protocols provided with the commercially available kits at the time of this chapter publication require only one organic extraction. This simple yet, as it turns out, quite useful modification allows access to previously inaccessible material. Potential benefits from these changes are a more comprehensive sample profiling of small RNA, as well as wider access to small volume samples, such as is typically available for acellular biofluids, which now can be prepared for small RNA sequencing on the Illumina platform.