Quantitation of alternatively spliced estrogen receptor alpha mRNAs as separate gene populations.
Estrogen receptor (ER) mRNA undergoes alternative splicing generating transcripts that have deletions in various combination of exons. Although several reports have shown that the spliced variant mRNAs are expressed in both normal and malignant tissues, the exact functional role(s) of these molecules have not been established in estrogen induced signal transduction processes mainly due to practical limitations involved in their detection and quantitation. We have recently described a 'Splice Targeted Primer Approach' that can specifically detect splice variants without amplifying the wild type ERs [12]. In the current report, we describe strategies to quantify individual splice variant mRNAs as separate gene populations independent of wild type or other variants using ER exon 7Delta and exon 2Delta as models. We describe the methods of quantifying the exon 7Delta and exon 2Delta transcripts in two breast cancer cell lines, MCF-7 and LCC2, and a breast tumor using the splice-targeted primers in combination with template competition RT PCR. The exon 2Delta splice specific sense primer along with an anti-sense primer in exon 4 amplified a 412 bp product in both cell lines and the tumor that could be quantitated. The exon 7Delta splice targeted anti-sense primer along with a partner primer in exon 2 amplified four transcripts that have deletions in exon 7, exons 7 and 4, exons 7 and 3-4, and exons 7 and 3-5. These four transcripts could be simultaneously quantified by the template competition method described here. Our results also show that the estrogen-independent LCC2 cells express significantly higher levels of the above 7Delta transcripts compared to the estrogen-dependent MCF-7 cells.
Koduri
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Department of Pharmacology, Howard University School of Medicine, 520 W. Street, NW, Washington, DC 20059, USA.
Poola
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D017398:Alternative Splicing; D001943:Breast Neoplasms / Q000235:genetics; D017931:DNA Primers; D047628:Estrogen Receptor alpha; D005091:Exons; D005260:Female; D018628:Gene Dosage; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis*; D011960:Receptors, Estrogen / Q000032:analysis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D047628:Estrogen Receptor alpha; D012333:RNA, Messenger; D011960:Receptors, Estrogen