Optimization and Standardization of Circulating MicroRNA Detection for Clinical Application: The miR-Test Case.
Clin Chem
; 62(5): 743-54, 2016 05.
Article
em En
| MEDLINE
| ID: mdl-27127244
ABSTRACT
BACKGROUND:
The identification of circulating microRNAs (miRNAs) in the blood has been recently exploited for the development of minimally invasive tests for the early detection of cancer. Nevertheless, the clinical transferability of such tests is uncertain due to still-insufficient standardization and optimization of methods to detect circulating miRNAs in the clinical setting.METHODS:
We performed a series of tests to optimize the quantification of serum miRNAs that compose the miR-Test, a signature for lung cancer early detection, and systematically analyzed variables that could affect the performance of the test. We took advantage of a large-scale (>1000 samples) validation study of the miR-Test that we recently published, to evaluate, in clinical samples, the effects of analytical and preanalytical variables on the quantification of circulating miRNAs and the clinical output of the signature (risk score).RESULTS:
We developed a streamlined and standardized pipeline for the processing of clinical serum samples that allows the isolation and analysis of circulating miRNAs by quantitative reverse-transcription PCR, with a throughput compatible with screening trials. The major source of analytical variation came from RNA isolation from serum, which could be corrected by use of external (spike-in) or endogenous miRNAs as a reference for normalization. We also introduced standard operating procedures and QC steps to check for unspecific fluctuations that arise from the lack of standardized criteria in the collection or handling of the samples (preanalytical factors).CONCLUSIONS:
We propose our methodology as a reference for the development of clinical-grade blood tests on the basis of miRNA detection.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
MicroRNAs
/
Reação em Cadeia da Polimerase em Tempo Real
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
/
Screening_studies
Limite:
Humans
Idioma:
En
Revista:
Clin Chem
Assunto da revista:
QUIMICA CLINICA
Ano de publicação:
2016
Tipo de documento:
Article