Quantitation of alternatively spliced estrogen receptor alpha mRNAs as separate gene populations

Estrogen receptor (ER) mRNA undergoes alternative splicing generating trans cripts that have deletions in various combination of exons. Although severa l reports have shown that the spliced variant mRNAs are expressed in both n ormal and malignant tissues, the exact functional role(s) of these molecule s have not been established in estrogen induced signal transduction process es mainly due to practical limitations involved in their detection and quan titation. We have recently described a 'Splice Targeted Primer Approach' th at can specifically detect splice variants without amplifying the wild type ERs [12]. In the current report, we describe strategies to quantify indivi dual splice variant mRNAs as separate gene populations independent of wild type or other variants using ER exon 7 Delta and exon 2 Delta as models. We describe the methods of quantifying the exon 7 Delta and exon 2 Delta tran scripts 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 2 Delta splice specific sense primer along with an anti-s ense primer in exon 4 amplified a 412 bp product in both cell lines and the tumor that could be quantitated. The exon 7 Delta splice targeted anti-sen se 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 t emplate competition method described here. Our results also show that the e strogen-independent LCC2 cells express significantly higher levels of the a bove 7 Delta transcripts compared to the estrogen-dependent MCF-7 cells. (C ) 2001 Elsevier Science Inc. All rights reserved.