On column purelink dnase

To assess the potential benefit of mRNA-stabilising reagents, we added TRIzol reagent (Life Technologies) to the venoms at a ratio of 5:1 as soon after venom extraction as safety permitted. RNA was isolated from the venom using the TRIzol Plus RNA Purification System following the manufacturer’s instructions (unless stated otherwise). To assess the effect of storage time and temperature on venom RNA stability, a sample of the B. arietans venom/TRIzol mixture was aliquoted into three equal volumes. Each sample was incubated at one of three temperatures: 4°C, 19°C or 37°C and RNA isolated (as per above) after 8, 24 or 48 hours. A sample of venom was also conventionally lyophilized and stored at 4°C until required. Each aliquot equated to 500 μL of liquid venom (equivalent to 124.66 mg lyophilized venom). All other venom samples had TRIzol added at a ratio of 5:1 and were processed using the TRIzol Plus RNA Purification System. All samples were DNase treated on column during the TRIzol Plus purification system protocol (On-Column PureLink DNase; Life Technologies) and total RNA was eluted in 30 μL nuclease free water (QIAGEN).

RNA from venom glands provided both the control for the venom RNA samples and the source for the transcriptomes from which the C-type lectin transcripts were assembled. Bitis arietans venom gland tissue (272 mg) was immediately homogenised under liquid nitrogen using a pestle and mortar and then a TissueRuptor (QIAGEN). Samples were DNase treated (On-Column PureLink DNase; Life Technologies) and total RNA was extracted and eluted in 30 μL nuclease free water (QIAGEN) using the TRIzol Plus RNA Purification System protocol. Venom gland transcriptomes were sequenced, assembled and annotated as previously described (Sanger sequenced EST transcriptome [28 (link)]; second generation Illumina transcriptome [29 (link)]).