Human reference RNA samples were prepared in the same format used by the SEQC/MAQC-III Consortium and ABRF Initiative using ERCC spike-ins (Jiang et al. 2011 (link); Li et al. 2014 (link); SEQC/MAQC-III Consortium 2014 (link)). Briefly, Sample A was prepared by doping 50 µL of Universal Human Reference RNA at 1 µg/µL (Agilent) with 1 µL of ERCC ExFold Mix 1 (Thermo Fisher Scientific), and Sample B was prepared by doping 50 µL of Human Brain Reference RNA at 1 µg/µL (Life Technologies) with 1 µL ERCC ExFold Mix 2. Samples A and B were then mixed in 3:1 or 1:3 ratios to constitute Samples C and D, respectively. Each sample was evaluated for integrity by using an Agilent 2100 Bioanalyzer (RNA 6000 Nano Chip Total Eukaryote RNA Assay, RIN ≥8.2) and then aliquoted and stored at −80°C.
Protocol detail
Preparation of ERCC-spiked Human RNA Samples
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Other organizations : The University of Texas at Austin
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Variable analysis
independent variables
- Mixing ratio of Sample A and Sample B (3:1 or 1:3)
- RNA integrity (RIN ≥ 8.2)
- Preparation of Sample A (50 µL of Universal Human Reference RNA at 1 µg/µL doped with 1 µL of ERCC ExFold Mix 1)
- Preparation of Sample B (50 µL of Human Brain Reference RNA at 1 µg/µL doped with 1 µL of ERCC ExFold Mix 2)
- RNA integrity evaluation using Agilent 2100 Bioanalyzer (RNA 6000 Nano Chip Total Eukaryote RNA Assay)
- Storage conditions (-80°C)
- ERCC ExFold Mix 1 (added to Sample A)
- ERCC ExFold Mix 2 (added to Sample B)
- Not explicitly mentioned
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