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Site-Directed Mutation and PCR Mimics 205
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Quantitative PCR for cAMP RI Alpha mRNA
Use of Site-Directed Mutation and PCR Mimics
John M. S. Bartlett
1. Introduction
Precise and accurate determination of mRNA expression levels in tissues and model
systems is a central methodology in a wide range of research applications. Expression
of many genes is currently assessed by northern blotting, RNAse protection assays,
Serial Analysis of Gene Expression (SAGE), and many other techniques; however, for
single transcripts, especially where tissue is limited or abundance is low, quantitative
polymerase chain reaction (PCR) is the method of choice. However, where quantitative
PCR is to be used, the reproducibility, accuracy, and detection limits of the technique
must be clearly defined.
We address this issue in the context of breast cancer by establishing a quantitative
PCR technique for the measurement of cAMP RI alpha-binding proteins, the regulatory
subunitis of cAMP-dependent protein kinase, using PCR mimics. The technique was
evaluated for interassay and intraassay variation and precision. This approach is
applicable to any marker of interest where quantitation of RNA or DNA levels by
PCR is attempted. For details of the construction of the mimics. see Bartlett et al. (1);
however, a more robust and reproducible method of mimic construction is described
within this volume (see Chapter 7).
2. Materials
1. RI alpha PCR primers (100 µM, see Note 1).
RI alpha 430 sense: 5′-GCATAACATTCAAAGCACTGC-3′
RI alpha antisense: 5′-CTTGCTGAATCACAGTCTCTCC-3′
2. RI alpha control (430 base pairs) in pCRII vector: Dilute the control plasmid to 100, 10,
1, and 0.1 pg of insert per µL (see Note 2).
3. RI alpha MIMIC (430 base pairs) in pCRII vector (see Note 3): Dilute the mimic plasmid
to 100, 10, 1, and 0.1 pg of insert per µL.
4. Extracted RNA (1 µg in 5 µL per sample; see Subheadings 2.1.3.–2.1.5., ibid).
5. Random hexamer N6 (100 µM, Life Technologies, Paisley, UK, see Note 4).
6. dNTP mix: 2 mM each of dATP, dTTP, dCTP and dGTP in distilled water, aliquoted, and
stored at –20°C (Life Technologies, Paisley, UK).
7. MMLV Reverse transcriptase and buffer (200 units/µL; Life Technologies, Paisley, UK).
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
205