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Extraction of RNA and Analysis of Estrogen-Responsive Genes by RT-qPCR

Michelle L Booze¹, Kathleen M Eyster²

¹Sanford Fertility and Reproductive Medicine, Sioux Falls, SD, USA. Michelle.Booze@SanfordHealth.org.

Methods in Molecular Biology (Clifton, N.J.)

|February 4, 2022

Abstract:

Reverse transcription-quantitative RT-PCR (RT-qPCR) is a powerful tool for assessing gene transcription levels. The technique is especially useful for measuring estrogen receptor transcript levels as well as gene expression changes in response to estrogen stimulation as it is quick, accurate, and robust and allows the measurement of gene expression in a variety of tissues and cells. This chapter describes the protocols used for RNA extraction and analysis as well as for RT-qPCR assay using hydrolysis (TaqMan-type) probes.

Keywords:

Custom design Estrogen receptors Fluorogenic probe Gene expression Master mix Normalization Primer RNA extraction RT-qPCR

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
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