Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5331

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In Vitro Transcription Using HeLa Cell Extracts and Primer Extension

Michael F. Carey, Craig L. Peterson, and Stephen T. Smale

Adapted from Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, 2nd edition, by Michael F. Carey, Craig L. Peterson, and Stephen T. Smale. CSHL Press, Cold Spring Harbor, NY, USA, 2009.


INTRODUCTION

Primer extension is a popular method for measuring transcription. Among the methods for start-site mapping in vivo, it is the one most easily adapted for in vitro transcription. Typically, DNA templates bearing activator binding sites are co-incubated with nuclear extracts (which provide the general transcription factors) and the corresponding activators to synthesize mRNA. In some cases, the activator is present in the extract (e.g., Sp1) and templates lacking or containing sites are compared. In the primer extension assay, a 32P-end-labeled DNA oligonucleotide is annealed to a complementary region in the mRNA and then extended by a reverse transcriptase to the 5' end of the mRNA. The resulting labeled cDNA is then fractionated and detected on a polyacrylamide/urea gel. The amount of the product is a measure of the transcriptional activation by the activators. Generally, the addition of 0.1 pmol of primer to the extension reaction is sufficient because it will be in vast excess over the amount of mRNA, facilitating quantitative primer hybridization and extension.


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M. F. Carey, C. L. Peterson, and S. T. Smale
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