External RNA controls consortium
The External RNA Control Consortium (ERCC) is an ad-hoc group with approximately 70 members from private, public, and academic organizations. The group was initiated in 2003 to develop a set of external RNA control transcripts that can be used to assess technical performance in gene expression assays. The external RNA controls will be added after RNA isolation, but prior to cDNA synthesis. They are being designed to evaluate whether the results for a given experiment are consistent with defined performance criteria. All ERCC work is intended to apply to quantitative, real-time reverse transcriptase polymerase chain reaction (QRT-PCR) assays as well as one-color and two-color microarray experiments.
The ERCC has worked together to define the desired properties of the transcripts, general protocols for their application, and an analysis scheme for performance assessment. In December 2003, the group developed a specification document that was discussed and refined in a public workshop at the National Institute of Standards and Technology (NIST) [1
]. Protocols for the use of external RNA controls in clinical applications are included in the Molecular Methods 16-P document from the Clinical and Laboratory Standards Institute, and were developed in a formal, accredited, open, consensus forum including several ERCC members. The analysis approach was developed in a public workshop at NIST in June 2004, and is based upon the measurement of pooled transcripts at known concentrations.
In the past year, the ERCC has refined specifications, generated and collected control sequences, evaluated optimal polyadenylated (polyA) tail length and identified a path forward for access and distribution of the controls. We are now initiating the Testing Phase of the project as described in this document.
Purpose of this document
During ERCC Testing, 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 authoritatively test and qualify individual controls. Qualification of a control sequence is consistent with the ISO 9000 definition of validation: "Confirmation, through the provision of objective evidence, that requirements for a specific intended use or application have been fulfilled." Based on the results of the test and qualification experiments, the ERCC will select a set of external RNA controls that perform consistently across technologies and platforms. DNA clones of the controls, basic informatics tools and appropriate documentation will be available to the public. Commercial products (e.g. primer sets and pools of transcripts) may also be developed and made available as Certified Reference Materials.
The ERCC is committed to open access and inclusive practices. We are distributing this description of the proposed testing process in an effort to gain consensus from the scientific community and to confirm the value of the final products. We hope it will be carefully reviewed by clinical and research laboratory scientists. On October 4–5, 2005, NIST hosted an ERCC Testing Workshop as an open forum to encourage feedback from the community and to invite volunteers to participate in the testing phase tasks. Comments from the meeting are posted on the NIST site [2
] and included in Appendix 2 of this document. Interested parties should contact Dr. Janet Warrington [3
] for further information.
The five specific aims of the testing project will result in production of a well-characterized, tested set of controls with demonstrated acceptable performance on major microarray platforms and commonly used QRT-PCR methods. The specific aims are as follows.
1. Design and produce the reagents necessary for the testing plan, including candidate external RNA controls, prototype arrays, primer sets for QRT-PCR and informatics tools for managing and annotating the testing results.
2. Qualify the reagents. Identify and minimize cross reactivity and potential interactions between the external controls, the probes used for their detection and background RNA molecules.
3. Qualify the assay by collecting performance data in multiple gene expression assays. Identify the linear range, sensitivity and reproducibility of individual candidate controls. Define performance criteria and select a candidate reference set of external RNA controls for future testing.
4. Qualify the product by using the candidate reference set of external RNA controls in typical microarray and QRT-PCR assays. Extend analyses to multiple RNA backgrounds and testing sites. Confirm the performance quality of the set of transcripts and the informatics tools used for their analysis. Finalize the reference set and ERCC products.
5. Distribute DNA clones of the external RNA controls and informatics tools to general scientific community. Publish report on ERCC materials, testing data and analysis methods. Write application use and general protocols.
Definitions, assumptions and limitations
In this document, the term "external RNA controls" refers to unlabeled, polyadenylated sense transcripts that are added to an RNA sample prior to processing and used to measure technical performance of the assay system. Although labeled transcripts will be generated during the testing effort to characterize the hybridization dynamics of each control, the final ERCC product is not intended to be used as a "hybridization control" in microarray experiments.
Use of external controls
The external RNA controls (sometimes referred to as "spikes") are intended to be added to total RNA samples before initiating the gene expression experiment. For microarray assays, a pool of multiple external RNA controls can be added to a single sample, then labeled and hybridized in parallel with the target. If each of the added spikes is introduced at a different concentration, a calibration curve can be constructed to indicate the linear range of the hybridization. Different pools of external RNA controls can be used in separate hybridizations (one-color system) or in different channels of the same hybridization (two-color system). For QRT-PCR assays, a single external RNA control can be added to the RNA sample and amplified simultaneously with the target sequence (two-color system) or independently in a replicate well (one-color system).
Current external RNA controls in microarray assays
Several sets of external RNA controls are currently available for microarray research [4
], but few probes corresponding to the controls are available on commercial microarrays and there is no consistency across platforms. As a result of the ERCC effort, a standard set of approximately 96 well-characterized, external RNA controls will be available. Many different array manufacturers are participating in the ERCC process and have committed to including probes complementary to the ERCC external RNA controls on their future microarray products.
Current external RNA controls in QRT-PCR assays
There are two RNA transcript quantification methods for QRT-PCR assays, absolute and relative quantification. Absolute quantification is very useful and accurate when QRT-PCR is used for measuring one or a few targets, but it often requires generation of template standards for each target and it may become cumbersome especially when many targets are analyzed [10
When QRT-PCR is used for gene expression profiling, or measuring dozens of transcripts simultaneously, relative quantification is often used. In this case, users rely on internal genes for normalization, usually a "housekeeping" gene or other transcript whose expression level is thought to be invariant. This approach has its own limitations. For example, an accurate relative comparison of gene expression requires similar primer affinities for both the target and the internal control, which are often difficult to achieve. Also, recent reports [12
] have shown that the expression levels of several housekeeping genes change in response to drug treatments or environmental changes, such as stress. These fluctuations raise questions about the general utility of a single universal set of genes as internal controls. Finally, the results of QRT-PCR reactions containing QRT-PCR inhibitors in RNA preparations are cumbersome to correct by using an internal gene transcript.
External RNA controls would provide an alternative method for normalization in relative quantification assays. The combination of external and internal or invariant controls is recommended as the most robust approach to optimal experimental control. Dose dependence, tissue specificity and degradation issues impact consistency of information and can be controlled for using a combination of controls. In addition, internal controls are more appropriately custom designed for analyte control in assays with a clearly defined intended use and therefore are by definition assay-dependent. The external RNA controls will provide a complementary resource to internal and assay-specific custom controls.
The ERCC product will be a set of clones that have been well characterized for performance on multiple microarray and QRT-PCR platforms. Candidate clones are submitted to the ERCC and evaluated as described in this document. Based on the testing results, the ERCC will select clones that perform acceptably on all participating platforms to be used as the reference set.
At this time, a number of organizations have submitted 140 candidate transcripts to be tested. As shown in Table , the candidate external RNA clones are either synthesized from unique sequences (i.e
., artificial) or are derived from genes in several non-mammalian species. Inserts are 500 – 2000 bp with a 20–30 bp polyadenylated tail. These clones are given freely without intellectual property rights. The ERCC welcomes additional submissions. Interested parties should contact Dr. Janet Warrington for further information [3
Summary of external RNA control clone library
This work culminates in the selection and distribution of materials that support the use of external RNA controls in expression assays. At the completion of the testing phase, the ERCC will release three products for use by the scientific community:
• DNA clones and sequences of the reference set of external RNA controls
• Basic informatics tools
• Publication of data, test results, protocols
Several additional reagents and documents will be developed during the testing phase. Many of the documents will be issued electronically to the general scientific community via NIST [2
] as supplemental information validating the control production process. Laboratory reagents will be used exclusively by ERCC members during testing. Some of the reagents will be donated by participating ERCC institutions, including array manufacturers, reagent manufacturers and NIST.