Quantitation of estrogen receptor mRNA and its alternatively spliced mRNAs in breast tumor cells and tissues.
Anal Biochem
; 245(2): 167-78, 1997 Feb 15.
Article
en En
| MEDLINE
| ID: mdl-9056208
ABSTRACT
Estrogen receptor (ER) mRNA exists as wild-type (full-length) and alternatively spliced variants in cell lines, normal tissues, and tumors. Most of the alternatively spliced variants discovered so far are missing one or more complete exons. RNase protection and RNA-PCR assays used previously to determine the relative concentration of a particular ER spliced-variant mRNA to wild-type mRNA have produced equivocal results because the probes/primers targeted only small regions within the nucleotide sequence. Variant ER mRNAs missing an exon outside the probe/primer region will react as if they were wild-type and any alternatively spliced variants containing a deletion at the probe/primer annealing site(s) will not be detected. A highly sensitive, competitive RNA-PCR assay has been developed that is quantitative with respect to the relative composition of wild-type ER and its alternatively spliced-mRNA forms, and semiquantitative with respect to their concentrations in cells and tissues. Separation and quantitation of the products are rapidly and accurately achieved by, respectively, capillary electrophoresis and laser-induced fluorescence. Wild-type ER mRNA concentration can be measured independently of all the reported exon deletion forms in a single PCR assay. Specific exon deletion forms can be measured by ER cDNA amplification with overlapping primer sets. Results obtained with RNAs isolated from two MCF-7 cell lines, a T-47D cell line, and five breast tumor tissues are presented.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Neoplasias de la Mama
/
ARN Mensajero
/
Receptores de Estrógenos
/
Reacción en Cadena de la Polimerasa
/
Empalme Alternativo
Tipo de estudio:
Diagnostic_studies
Límite:
Female
/
Humans
Idioma:
En
Revista:
Anal Biochem
Año:
1997
Tipo del documento:
Article
País de afiliación:
Estados Unidos