Regular Article
Real-Time PCR Quantification of Full-Length and Exon 11 Spliced BRCA1 Transcripts in Human Breast Cancer Cell Lines

https://doi.org/10.1006/bbrc.2000.3100Get rights and content

Abstract

Germline alterations of the BRCA1 tumor suppressor gene have been implicated at least in half of familial breast cancers. Nevertheless, in sporadic breast cancer no mutation of this gene has been characterized to date. In sporadic breast tumors, other BRCA1 gene loss of function mechanisms, such as down-regulation of gene expression, have been suggested. In an effort to better understand the relationship between BRCA1 expression and malignant transformation, we have adapted the new real-time quantitative PCR method based on a 5′ nuclease assay and the use of doubly labeled fluorescent TaqMan probes to quantify BRCA1 mRNA. We have compared expression of BRCA1 mRNA with or without exon 11 in the normal breast epithelial cell line MCF10a and in three cancer cell lines (MCF-7, MDA-MB231 and HBL100) by comparing two methods of quantification: the comparative CT and the standard curve. We found that the full length BRCA1 mRNA, which encodes the functional nuclear protein, was down-regulated in tumor cells when compared with MCF10a cells.

References (30)

  • D. Ford et al.

    Am. J. Hum. Genet.

    (1998)
  • R. Hakem et al.

    Cell

    (1996)
  • P.A. Futreal et al.

    Science

    (1994)
  • S.D. Merajver et al.

    Nat. Genet.

    (1995)
  • M.E. Thompson et al.

    Nat. Genet.

    (1995)
  • Y. Miki et al.

    Science

    (1994)
  • J.M. Gudas et al.

    Cell Growth Differ.

    (1996)
  • C.A. Wilson et al.

    Oncogene

    (1997)
  • P.M. Holland et al.

    Proc. Natl. Acad. Sci. USA

    (1991)
  • U.E. Gibson et al.

    Genome Res.

    (1996)
  • H.D. Soule et al.

    Cancer Res.

    (1990)
  • H.D. Soule et al.

    J. Natl. Cancer Inst.

    (1973)
  • R. Cailleau et al.

    J. Natl. Cancer Inst.

    (1974)
  • E.V. Gaffney et al.

    J. Natl. Cancer Inst.

    (1979)
  • Cited by (47)

    • Quantification of type II procollagen splice forms using alternative transcript-qPCR (AT-qPCR)

      2012, Matrix Biology
      Citation Excerpt :

      There are few practical methods for the routine quantitative analysis of alternative mRNA splice forms. Most commonly, PCR-based, co-amplification approaches, generating multiple PCR products have been used, with primers that flank alternative exon–exon junctions (Kafert et al., 1999; Dalski et al., 2000; Davis-Taber et al., 2000; Favy et al., 2000; Brown et al., 2004; Connell et al., 2005; Meidan et al., 2005; Yoong et al., 2005; Nygard et al., 2010; Long et al., 2011; Sylvestersen et al., 2011). Although convenient, the co-amplification approach, is semi-quantitative at best, and may under- or over-estimate relative quantities of alternative splice forms.

    • Genistein and daidzein act on a panel of genes implicated in cell cycle and angiogenesis by Polymerase Chain Reaction arrays in human prostate cancer cell lines

      2010, Cancer Epidemiology
      Citation Excerpt :

      Two independent reverse transcriptions were also performed for the RNA extractions. Each result was analyzed and expressed as a mean ± SD [24]. Each value represents the mean ± SD of three assays.

    • A novel substitution at the translation initiator codon (ATG → ATC) of the lipoprotein lipase gene is mainly responsible for lipoprotein lipase deficiency in a patient with severe hypertriglyceridemia and recurrent pancreatitis

      2006, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      Each cycle consisted of a 40-s denaturation at 94 °C, 40-s annealing at 58 °C, and a 60-s extension at 72 °C. The mRNA levels were normalized to 18S RNA using the comparative cycle threshold (CT) method [12]. LPL mass analysis.

    • A new alternative splice variant of BRCA1 containing an additional in-frame exon

      2005, Biochimica et Biophysica Acta - Gene Structure and Expression
    • Staphylococcal enterotoxin A modulates intracellular Ca<sup>2+</sup> signal pathway in human intestinal epithelial cells

      2005, FEBS Letters
      Citation Excerpt :

      Each cycle consisted of a 30-s (for eNOS and GAPDH) or 60-s (for iNOS) at 94 °C for denaturing, 30-s at 55 °C (eNOS and GAPDH) or 56 °C (iNOS) for annealing, and 120-s at 72 °C for extension. The mRNA levels were normalized to GAPDH by using the comparative cycle threshold (Ci) method [13]. Henle 407 cells cultured on glass cover slips were loaded with Fura-2/AM.

    View all citing articles on Scopus
    1

    To whom correspondence should be addressed. Fax: 33 (0)4 73 27 80 42. E-mail: [email protected].

    View full text