Additional file 1:

Performance of Nanostring spike-ins. Nanostring spike-ins span a reported range from 0.25 fM to 50 fM. All samples were assayed in triplicate; data are broken down by replicate. nCounter™ spike-ins consisted of two parallel sets of the same RNAs made into two different cocktails (cocktails #3 and #4). Some RNAs are set at different concentrations between these cocktails while others are at the same concentration (Table 3).

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Additional file 2:

Performance of Rosetta spike-ins. The Rosetta spike-ins were originally constructed to measure dynamic range, ratio fidelity, and hybridization quality of an in-house, two-color microarray system [4]. Data from the nCounter™ spike-in controls were used to derive the precision profile and to provide a calibration curve for quantitation of other RNA transcripts. Spike-ins curves showed good linearity and reproducibility, and good conservation of relative measurements. Overall, the precision of the platform for the Rosetta Spike-ins had CVs of approximately 6%, except for concentrations below 1.5 fM (CVs were 10–30%). Error bars represent standard deviations.

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Additional file 3:

Comparison of intensity values derived from Taqman^® (x-axis) and Nanostring (y-axis) for each of the four MAQC samples (UHR, 25% Brain/75% UHR, 75% Brain/25% UHR, and Brain). Markers in red represent genes that were outside of the calibration curve. Pair-wise nCounter™ to Taqman^® comparisons of measured expression levels of genes on a per sample basis showed correlation coefficients ranging from 0.835 to 0.886. Error bars represent standard deviations.

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Additional file 4:

Comparison of calculated log₂ ratios for Brain vs UHR for Nanostring and Taqman^®. Transcripts that were outside the calibration curve are shown in red and are labeled with their lowest measured concentration in either Brain or UHR (in fM). In the comparison of ratios, data for nCounter™ and Taqman^® were first corrected for the known differences in mRNA content between Brain and UHR (2% to 3%). Additional File 4 shows the fit of the ratio comparisons to the line y = x for samples whose intensity falls within the standard curve (blue squares in Additional File 4), R = 0.995, demonstrating good linear agreement between Taqman^® and nCounter™ ratio measurements.

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Additional file 5:

Data from Nanostring nCounter experiment. This spreadsheet contains the raw nCounter™ data, the Taqman^® data used for comparisons, and the normalized and averaged nCounter™ data.

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Additional file 6:

Representative Bioanalyzer traces from FFPE-derived RNA. These traces demonstrate the degree of RNA degradation assayed by the nCounter™ system in our experiments.

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