Prior to RNA/cDNA sequencing, it is common practice to perform quality control on the sample, using the Agilent 2100 Bioanalyzer to determine the RNA integrity number (RIN). The RIN assigns a measurement of the level of degradation of the RNA in the extract, from 1-10, with 10 being the least degraded. For more information, see the RNA Integrity Number (RIN) document from Agilent. It is recommended that RNA extracts have an RIN of seven or higher before proceeding with library preparation for nanopore sequencing, further explained in the RNA Integrity Number - RIN page.

We have investigated the stability of extracted RNA over time when stored at different temperatures and in different buffers. Total RNA was extracted from a GM12878 cell pellet using a TRIzol®-based extraction method and the RNA was either eluted in water, TE Buffer or RNA Storage Solution, aliquoted and stored at room temperature, 4°C or -20°C. After set periods of time, aliquots were removed from storage and the RIN was determined using the Agilent 2100 Bioanalyzer (Figure 1). We advise against storage of RNA samples in water at room temperature and to determine RIN prior to library preparation to ensure RIN is of seven or higher, to prevent short reads.

Figure 1. The effect of different storage conditions on the RIN of extracted total RNA.