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

被引:17
作者
Koduri, S
Poola, I
机构
[1] Howard Univ, Sch Med, Dept Pharmacol, Washington, DC 20059 USA
[2] Howard Univ, Sch Med, Dept Biochem & Mol Biol, Washington, DC 20059 USA
关键词
estrogen receptor alpha; splice variants; splice targeted primers; mRNA quantitation; template competition RT PCR;
D O I
10.1016/S0039-128X(00)00128-8
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 7 Delta and exon 2 Delta as models. We describe the methods of quantifying the exon 7 Delta and exon 2 Delta 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 2 Delta 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 7 Delta 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 7 Delta transcripts compared to the estrogen-dependent MCF-7 cells. (C) 2001 Elsevier Science Inc. All rights reserved.
引用
收藏
页码:17 / 23
页数:7
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