The SIRV sequences conform to the canonical exon-intron junction rule: 96.9 % of all SIRV junctions are GT-AG, with the less frequent variants being present at 1.7% (GC-AG) and 0.6% (AT-AC). Two non-canonical splice sites were included at 0.4% each (CT-AG and CT-AC).
You can download the sequences – SIRV sequence design overview (XLSX)
The SIRVs were produced by T7 transcription from synthetic genes which – while optimized – in some cases generates RNA of varying integrity. A series of tailored methods was applied to purify full-length SIRV RNAs with a minimal amount of any side products.
Synthetic gene constructs are produced (Bio Basic Inc, Markham, ON, Canada) that comprised 5’ to 3’ a unique restriction site, 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 that is fused with an exclusive 2nd restriction site. These gene cassettes are cloned into a vector, colony-amplified and singularized. All SIRV sequence in the purified plasmids are verified by Sanger-sequencing to identify the correct clones. The E. coli cultures are grown in batches to obtain plasmids in the lower µg-scale. Double digestion of isolated plasmids with XhoI and NsiI must show correct insert size and complete restriction. Linearized, silica-purified plasmids serve as templates in in vitro transcription reactions using T7 transcription kits (AmpliScribe T7 High Yield Transcription Kit, and AmpliScribe T7 Flash Transcription Kit, Epicentre, Madison, WI). The DNase-treated, phenol-extracted and silica-purified in vitro transcription products are assessed for concentration and purity by spectrophotometry (NanoDrop, Thermo Fisher Scientific, Waltham, MA) and for integrity by capillary electrophoresis (2100 Bioanalyzer, RNA 6000 Pico Kit, Agilent Technologies, Santa Clara, CA).
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 transcripts (average 1.1 kb with 14 RNAs between 2.0 and 2.5 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. A majority of transcripts must be purified by at least one of two purification methods. Purification method one is selective for poly(A)-tails and neglects prematurely terminated transcription products. Purification method two is based on size-selective quantitative electrophoresis separating the correctly sized main products from shorter fragments (transcription break off and degraded products) and longer fragments (run-through transcription products). After purification, the 69 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.
In contrast, the comprehensive and novel set of Spike-In Transcript Variants, SIRVs, can be used to validate isoform-specific RNA sequencing workflows and to compare experiments by extrapolating the results from the well-defined isoform ground truth of a small fraction of control reads to the sample reads. Within the context of variant detection assessment of dynamic range, dose response, lower limit of detection, and fold-change response is possible as well.
In another example: If 10 ng total RNA input are to be spiked, then 1 µl can be diluted 1:10000. See also Table 3, p.10 of the User Guide.
In any case we do not recommend to keep the dilution for very long as the diluted RNA solutions are increasingly unstable