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High‐Throughput Quantitative Real‐Time PCR

Zoltan P. Arany¹

¹Cardiovascular Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 11.10

DOI:

10.1002/0471142905.hg1110s58

Online Posting Date:

July, 2008

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Abstract

Recent technical advances in quantitative real-time PCR (qRT-PCR) have allowed for extensive miniaturization, thereby rendering the technique amenable to high-throughput assays. Large numbers of different nucleic acids can now rapidly be measured quantitatively. Many investigations can benefit from this approach, including determination of gene expression in hundreds of samples, determination of hundreds of genes in a few samples, or even quantification of nucleic acids other than mRNA. A simple technique is described here to quantify 1880 transcripts of choice from any number of starting RNA samples. Curr. Protoc. Hum. Genet. 58:11.10.1–11.10.11. © 2008 by JohnWiley & Sons, Inc.

Keywords: quantitative real-time PCR; qRT-PCR; high-throughput; gene expression

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Introduction
Strategic Planning
Basic Protocol: High-Throughput qPCR Screening
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol: High-Throughput qPCR Screening

Materials

Samples of interest: e.g., untreated (control) and treated samples
RNA isolation kit (e.g., Trizol, Invitrogen; RNeasy, Qiagen)
cDNA preparation kits with reverse transcription reagents (e.g., iScript cDNA synthesis kit, Bio-Rad)
RNAse/DNAse-free water (e.g., commercially available from Integrated DNA Technologies)
Stock plates of oligomers designed to amplify the transcripts of interest (e.g., from Integrated DNA Technologies), including four internal controls (e.g., HPRT, TBP, 18S, and G3PD)
2× SYBR mix containing enzyme, buffer, dNTPs (e.g., ABI)

Thermal cycler
Standard size deep-well 384-well plates
qPCR-compatible 384-well plates (e.g., ABI; depends on qPCR reader used)
Robotic liquid handling equipment (e.g., Multidrop Combi dispenser, Thermo Scientific; CyBi-Well Vario, CyBio)
Racks of tips for 96-well and 384-well robotic liquid transfers (e.g., CyBio)
Optical plate seals (e.g., ABI)
Centrifuge with rotor adapted for microtiter plates
ABI Prism 7900 real-time qPCR instrument (or similar; see Strategic Planning)

Additional reagents and equipment for performing gel electrophoresis of RNA (see appendix 3K)

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Figures

Figure 11.10.1

Sample amplification curves seen after performing qPCR on a single cDNA sample with 384 different oligo primer pairs, in a single 384-well plate. See text for details.

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Figure 11.10.2

Ideal (A) and poor (B) dissociation curves for the amplification product from different pairs of oligos. See text for details. Note that the y axis denotes the derivative of the change in fluorescence—i.e., not the fluorescence itself, but rather how fast the amplification products are denaturing at a given temperature.

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Literature Cited

Literature Cited
	Mullis, K. 1990. The unusual origin of the polymerase chain reaction. Scientific American 2624:56-61, 64-65.
Key References
	Ding, C. and Cantor, C.R. 2004. Quantitative analysis of nucleic acids—the last few years of progress. J. Biochem. Mol. Biol. 37:1-10.
	Lutfalla, G. and Uze, G. 2006. Performing quantitative reverse-transcribed polymerase chain reaction experiments. Methods Enzymol. 410:386-400.