In brief
The three SIRV Isoform Mixes (E0, E1 and E2) are each composed of 4 SIRV transcript SubMixes.
These four SubMixes are prepared from one of 8 PreMixes, allowing for the unique identification of each SIRV by capillary electrophoresis while entering the mixing scheme (Paul et al. , 2016).
In detail
The four pairs of PreMixes are combined in equal ratios to SubMixes, before those SubMixes are combined in defined ratios. The combination of accurate volumes of the stock solutions in sufficiently large batch sizes and a transparent monitoring of the sequential processing steps warrant the most accurate preparation of the mixes above process inherent lower boundaries.
Pipetting errors vary depending on transfer volumes and range from ±4 % for 2 µl to ±0.8 % for >100 µl transfers.
The precision was experimentally determined with blank solutions. Starting with the stock concentration measurement (NanoDrop accuracy ±2 ng/µl for 50 ng/µl), and accounting for the entire mixing pathway, the accumulative concentration error is expected to range between ±8 % and ±4.7 %. Therefore, in the data evaluation one has to account for the experimental variability by allowing for lower accuracy thresholds of ±8 % on the linear scale, or ±0.11 on the log2-fold scale, respectively.
The SIRV concentration ratios between two mixes are more precise because only one final pipetting step defines the concentration differences and synchronizes all SIRVs, which belong to the same SubMix. Here, a maximal error of ±4 % (or ±0.057 on the log2-fold scale) can be expected between SubMixes, while all SIRVs of the same SubMix must propagate coherently into the final mixes.
Bioanalyzer traces are used to monitor the relative propagation of the SIRVs, PreMixes and SubMixes during the mixing. In addition, the accurate pipetting of the 8 PreMixes is controlled by checksums of Nanodrop concentration measurements by weighing on an analytical balance
An Illustration of how the SubMixes contribute to SIRV transcript concentrations within each Isoform Mix is illustrated in the SIRV-Set 1 User Guide.