Optimized protocol for gene expression analysis in formalin-fixed, paraffin-embedded tissue using real-time quantitative polymerase chain reaction.
Diagn Mol Pathol
; 18(3): 176-82, 2009 Sep.
Article
en En
| MEDLINE
| ID: mdl-19704263
ABSTRACT
Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common tissue specimen available after microscopic examination. Molecular methods, such as polymerase chain reaction (PCR) and gene expression examination, serve as a source of diagnostic and prognostic information but require high-quality RNA. However, the increasing application of RNA extracted from FFPE tissue frequently results in very small and degraded quantities of nucleic acid. This study targets gene expression analysis from FFPE specimens using real-time quantitative PCR. The whole protocol consists of several steps, that is, RNA extraction and its quality control, reverse transcription, and fluorescence detection during real-time quantitative PCR. We compared several methods in each step, chose the most effective, and with that combination we successfully examined 95% (62 from 65) FFPE samples for our genes of interest. We reached the best results with RNA isolation by using a commercial kit, carefully interpreted UV spectrophotometric values, and meticulously chose reverse transcriptase and TaqMan fluorescence detection. Our protocol improves the utility of FFPE tissue for molecular profiling studies.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Patología
/
Manejo de Especímenes
/
Reacción en Cadena de la Polimerasa
/
Perfilación de la Expresión Génica
Tipo de estudio:
Evaluation_studies
/
Guideline
Límite:
Humans
Idioma:
En
Revista:
Diagn Mol Pathol
Asunto de la revista:
BIOLOGIA MOLECULAR
/
PATOLOGIA
Año:
2009
Tipo del documento:
Article
País de afiliación:
República Checa