Evaluation of the External RNA Controls Consortium (ERCC) reference material using a modified Latin square design.
BMC Biotechnol
; 16(1): 54, 2016 Jun 24.
Article
en En
| MEDLINE
| ID: mdl-27342544
ABSTRACT
BACKGROUND:
Highly multiplexed assays for quantitation of RNA transcripts are being used in many areas of biology and medicine. Using data generated by these transcriptomic assays requires measurement assurance with appropriate controls. Methods to prototype and evaluate multiple RNA controls were developed as part of the External RNA Controls Consortium (ERCC) assessment process. These approaches included a modified Latin square design to provide a broad dynamic range of relative abundance with known differences between four complex pools of ERCC RNA transcripts spiked into a human liver total RNA background.RESULTS:
ERCC pools were analyzed on four different microarray platforms Agilent 1- and 2-color, Illumina bead, and NIAID lab-made spotted microarrays; and two different second-generation sequencing platforms the Life Technologies 5500xl and the Illumina HiSeq 2500. Individual ERCC controls were assessed for reproducible performance in signal response to concentration among the platforms. Most demonstrated linear behavior if they were not located near one of the extremes of the dynamic range. Performance issues with any individual ERCC transcript could be attributed to detection limitations, platform-specific target probe issues, or potential mixing errors. Collectively, these pools of spike-in RNA controls were evaluated for suitability as surrogates for endogenous transcripts to interrogate the performance of the RNA measurement process of each platform. The controls were useful for establishing the dynamic range of the assay, as well as delineating the useable region of that range where differential expression measurements, expressed as ratios, would be expected to be accurate.CONCLUSIONS:
The modified Latin square design presented here uses a composite testing scheme for the evaluation of multiple performance characteristics linear performance of individual controls, signal response within dynamic range pools of controls, and ratio detection between pairs of dynamic range pools. This compact design provides an economical sample format for the evaluation of multiple external RNA controls within a single experiment per platform. These results indicate that well-designed pools of RNA controls, spiked into samples, provide measurement assurance for endogenous gene expression studies.Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ARN
/
Análisis de Secuencia de ARN
/
Perfilación de la Expresión Génica
/
Secuenciación de Nucleótidos de Alto Rendimiento
Tipo de estudio:
Diagnostic_studies
/
Evaluation_studies
Límite:
Humans
Idioma:
En
Revista:
BMC Biotechnol
Asunto de la revista:
BIOTECNOLOGIA
Año:
2016
Tipo del documento:
Article
País de afiliación:
Estados Unidos