Human brain reference rna

We purchased Universal Human Reference RNA from Agilent Technologies, Inc., and Human Brain Reference RNA from Life Technologies, Inc. For the nCounter experiments, we used the same RNA sample types as the SEQC consortium. We assessed RNA purity and integrity with Bioanalyzer (Agilent Technologies, Inc.) prior to use in the nCounter assays. Sample preparation and analysis were done using a nCounter Prep Station 5 s and a nCounter Digital Analyzer 5s. Expression of 770 genes (~730 genes with ~40 housekeeping genes and positive and negative controls) was assessed using the nCounter Human PanCancer Pathways Panel. A second PanCancer Pathways Panel was run using the same samples submitted to the first panel to assess the effect of batches on nCounter results. We used the NanoStringNorm function^{45 (link)} in the R NanoStringNorm package (https://cran.r-project.org/web/packages/NanoStringNorm/index.html) to normalize the dataset. Boxplots and MDS plots were used to confirm the samples were properly normalized and to check for the presence of batch effects. The two-sided Wilcoxon rank sum test was used to compare the distribution of the fold-change between long and short genes across the three different platforms—RNA-seq, Microarray and NanoString.

The three total human RNA sources distributed were obtained from Ambion (Thermo Fisher Scientific): Human Brain Reference RNA (Cat. No. AM6050), Human Liver Total RNA (Cat. No. AM7960) and Human Placenta Total RNA (Cat. No. AM7950) Two concentrations of the total RNA were used in each library preparation. RNA was quantified in triplicate using Quant-iT Ribogreen RNA Assay kit, Low-Range protocol (R11490; ThermoFisher). We used 1 μg, which is the staring input recommended by each sequencing kit, and 10 ng. A large pool of plasma (30 mL) was used for comparison of kits and input volumes. Whole blood was collected from normal healthy volunteers in EDTA tubes, spun at 2500 rpm to separate plasma. Plasma was pooled, and 1 mL aliquots placed in tubes and stored at − 80 °C. The appropriate number of tubes were combined and isolated for each comparison. For example, for the 5 mL input volume, 5 aliquots were selected and the RNA isolated (using miRVana Paris, ThermoFisher AM1556, with modifications [25 ] and sample preparation for sequencing performed. The samples were collected in accordance with established Institutional Review Board protocols (WIRB #20142635).

ERCC standards and Human Brain Reference RNA were purchased from Thermo Fisher Scientific (4456739 and AM6050, respectively). Universal Human Reference RNA was purchased from Agilent Technologies (740000) and resuspended in RNA Storage Solution (Thermo Fisher Scientific AM7001), which was also used for all RNA dilutions. HBRR and UHRR stocks were measured with a Quant-iT RNA Assay Kit (Thermo Fisher Scientific Q10213) on a Qubit fluorometer, and these concentrations were used to calculate dilutions. All RNA stocks were stored at −80°C.

For this benchmark we used the well-characterized MAQC-I RNA-samples MAQCA (Universal Human Reference RNA, Agilent Technologies,) and MAQCB (Human Brain Reference RNA, Thermo Fisher Scientific)¹⁴. For both samples, RNA-sequencing was performed.