General guidelines, how to prepare your RNA/DNA samples for NGS

Preparation of RNA samples

Extract your RNA with preferred kit (e.g. Qiagens RNeasy Mini Kit (Catalog no. 74104) with Optional On-Column DNase Digestion with the RNase-Free DNase Set (cat. no. 79254)). Please, be sure that your RNA samples are free from DNA, as this will result in an underestimation of the amount of RNA used.
 
From extracted RNA, three aliquots should be separated before freezing to avoid extra freezing-thawing cycles. These aliquots need to present the sample-aliquots to be sequenced, so no dilutions, or additional freezings for these.

  • First aliquot (preferably 8 µl/sample) is for measuring precise concentration of samples with Qubit-assay.
  • The second aliquot (2 µl/sample) is for Bioanalyzer-assay for determining the RNA Integrity Number (RIN)-values.
  • The third aliquot (15-20 ul) is used for library preparation.

Please contact to ngs (at) oulu.fi for any questions, or if you have only limited amount of RNA.

Preparation of DNA samples

The purity and approximate concentration should be measured for all samples (e.g. NanoDrop). The recommended thresholds are:

  • 260/280 value 1.7-2, preferably 1.8
  • 260/230 value > 1.5, preferably > 1,8

Please send this information (purity/concentration) to ngs (at) oulu.fi, to receive instructions how to proceed.

Last updated: 3.12.2018