High‐Throughput Quantitative Real‐Time PCR

Zoltan P. Arany1

1 Cardiovascular 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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: High‐Throughput qPCR Screening
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: 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 )
  • Additional reagents and equipment for performing gel electrophoresis of RNA (see appendix 3K)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library