In short
The SIRVs isoforms and long SIRVs are produced by T7 transcription from synthetic genes, and a series of tailored methods was applied to purify full-length SIRV RNAs with a minimal amount of side products.
In detail
Synthetic gene constructs were obtained that comprised 5' to 3' a T7 RNA polymerase promoter whose 3' G is the first nucleotide of the actual SIRV sequence, which is seamlessly followed by a (A30)-tail. These gene cassettes are cloned into a vector, colony-amplified and singularized. All SIRV sequences in the purified plasmids are verified by Sanger-sequencing to identify the correct clones. Linearized, silica-purified plasmids then serve as templates in in vitro transcription reactions using T7 transcription kits The DNase-treated, phenol-extracted and silica-purified in vitro transcription products are assessed for concentration and purity by spectrophotometry and for integrity by capillary electrophoresis. In the context of variant verification RNA integrity is a very important measure. Fragments arising from incomplete transcription might impose errors on the correct determination of variants which share those sequences and thereby also affect the overall gene coverage.
The integrity of the transcription products is very heterogeneous as expected, given the broad sequence variation and the length of the SIRV isoform transcripts (between 191 bp and 12 kb). Therefore, a set of tailored purification procedures is applied to obtain full-length RNAs with a minimal amount of side products despite the broad sequence and length variation of the SIRVs.
After purification, SIRV RNAs are assessed for the ratios of pre-peak fraction, main-peak fraction (corresponding to RNAs of correct length), and post-peak fraction. Finally, the SIRVs are quantified by absorbance spectroscopy to adjust all stock solutions to a base concentration of close to, but above 50 ng/µl, and to monitor RNA purity by absorbance ratio of 260/280 nm, and 260/230 nm.