Proposed methods for testing and selecting the ERCC external RNA controls

Abstract

The External RNA Control Consortium (ERCC) is an ad-hoc group with approximately 70 members from private, public, and academic organizations. The group is developing a set of external RNA control transcripts that can be used to assess technical performance in gene expression assays. The ERCC is now initiating the Testing Phase of the project, during which candidate external RNA controls will be evaluated in both microarray and QRT-PCR gene expression platforms. This document describes the proposed experiments and informatics process that will be followed to test and qualify individual controls. The ERCC is distributing this description of the proposed testing process in an effort to gain consensus and to encourage feedback from the scientific community. On October 4-5, 2005, the ERCC met to further review the document, clarify ambiguities, and plan next steps. A summary of this meeting and changes to the test plan are provided as an appendix to this manuscript.

Figure 1

Illustrations of latin square and graeco-latin square designs. "A1" to "A4" number the 4 arrays used in the experiment, "G1" to "G4" number the 4 transcripts being studied and "L1" to "L4" denote 4 different concentrations for each transcript. The four pools of transcripts are labeled "W" to "Z". "g" and "r" note the gene concentrations or pools used in the green or red channel, respectively of a two-color experiment.

Figure 2

Illustration of chi square fit. Panel A. The distances from a straight-line fit (arrows) are calculated. Panel B. The Chi square fit of the distances is then determined.

Figure 3

Illustration of spike performance.

Figure 4

Illustration of model data (including modeled noise). The values of m and b that were input into the model were m = 0.85 and b = 0.08. The noise model is realistic, in that it includes both constant (scanner) and proportional (chemical) noise.