High‐Throughput RT‐PCR for Small‐Molecule Screening Assays

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Quantitative measurement of the levels of mRNA expression via real?time reverse transcription polymerase chain reaction (RT?PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real?time PCR analysis, as well as the commercial availability of start?to?finish kits for RT?PCR, has enabled the use of this method for high?throughput small?molecule screening on a scale comparable to traditional high?throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT?PCR as a primary small?molecule screening assay, including the different methods available for mRNA isolation and analysis.Curr. Protoc. Chem. Biol. 4:49?63 © 2012 by John Wiley & Sons, Inc.

Keywords: real?time PCR; high?throughput screening; phenotypic screening; qRT?PCR; gene expression

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Introduction
Strategic Planning
Basic Protocol 1: Two‐Step cDNA Generation and qPCR Analysis
Alternate Protocol 1: Generation of cDNA Using Oligo‐dT Capture Plates
Alternate Protocol 2: One‐Step qRT‐PCR
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Two‐Step cDNA Generation and qPCR Analysis

Materials

Adherent cells of interest
Small molecule compounds to be screened
Vehicle: dimethylsulfoxide (DMSO)
Phosphate‐buffered saline (PBS), pH 7.4
RNA isolation and cDNA preparation kit (e.g., Applied Biosystems Cells‐to‐Ct, Qiagen Fastlane, Roche RealTime Ready Cell Lysis and Transcriptor cDNA kit) containing:
- Lysis solution (with DNase I)
- Stop solution (to stop DNase I reaction)
- Reverse transcriptase (RT) buffer
- Reverse transcriptase (RT) enzyme
Nuclease‐free H₂ O
Real‐time qPCR master mix containing dNTPs, buffer, polymerase (with or without SYBR Green depending on detection method; e.g., Roche SYBR Green master mix or Roche Probes master mix)
20× primer set (if SYBR Green master mix is used) or probes/primer set (if Roche Probes master mix is used), for each gene to be analyzed (Roche)

Multiwell sterile cell culture–treated plates
Tissue culture incubator (37°C, 5% CO₂ , 95% humidity)
Multichannel pipettor or robotic pipetting station (e.g., CyBio Vario)
Bulk reagent dispenser (e.g., Thermo Combi Multidrop) and accessories
Plate washer (e.g., Biotek EX‐405; optional)
Optional: Acoustic dispenser (Labcyte Echo or EDC ATS‐100) and acoustic‐certified source plates
Multiwell RNase‐free PCR plates
Sealing film for PCR plates
Centrifuge for multiwell plates
Standard multiwell PCR block (optional)
Multiwell qPCR plates
Optical sealing film for qPCR plates
RNase‐free tips, either for multichannel pipettor or robotic pipetting station
Real‐time qPCR instrument (see Strategic Planning)

Additional reagents and equipment for cell culture (Phelan, )

Alternate Protocol 1: Generation of cDNA Using Oligo‐dT Capture Plates

Turbocapture cDNA kit (Qiagen, cat. no. 72271 for 384‐well, 72251 for 96‐well plates)
Buffer TCL (Qiagen, cat. no. 1031586)

Alternate Protocol 2: One‐Step qRT‐PCR

Adherent cells of interest
One‐step qPCR kit (e.g., Roche Real‐time Ready cell lysis kit, cat. no. 05943523001)

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Figures

Figure 1. Three alternative protocols for qRT‐PCR depending on cell type.

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Figure 2. Amplification curves from one channel of a two‐color 384‐well PCR experiment. Anomalously shaped curves (one shown) or nonamplifying curves (none shown) can be discarded or assigned arbitrarily high C_q values for downstream calculations.

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Figure 3. Plotting of concentration‐response curve of hit compounds for reduced expression of a target gene. By converting to the relevant biological measurement, fold change, the apparent EC₅₀ has shifted approximately 2‐fold. Hypothetical data shown in Table .

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Figure 4. Construction of a standard curve to measure PCR efficiency. An arbitrary starting amount of template (in this case, sufficient to give a C_q of 15) is 2‐fold serially diluted and the dilution series is measured by real‐time qPCR. The slope of the resulting C_q values versus −log₂ of the dilution is related to the PCR efficiency by the equation Eff = 2^1/slope . Idealized curves are shown.

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Videos

Literature Cited

	Arany, Z. 2008. High‐throughput quantitative real‐time PCR. Curr. Protoc. Hum. Genet. 58:11.10.1‐11.10.11.
	Arany, Z., Wagner, B.K., Ma, Y., Chinsomboon, J., Laznik, D., and Spiegelman, B.M. 2008. Gene expression‐based screening identifies microtubule inhibitors as inducers of PGC‐1 alpha and oxidative phosphorylation. Proc. Natl. Acad. Sci. U.S.A. 105:4721‐4726.
	Arikawa, E., Sun, Y., Wang, J., Zhou, Q., Ning, B., Dial, S.L., Guo, L., and Yang, J. 2008. Cross‐platform comparison of SYBR Green real‐time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC) study. BMC Genomics 9:328.
	Benes, V. and Castoldi, M. 2010. Expression profiling of microRNA using real‐time quantitative PCR, how to use it and what is available. Methods 50:244‐249.
	Derveaux, S., Vandesompele, J., and Hellemans, J. 2010. How to do successful gene expression analysis using real‐time PCR. Methods 50:227‐230.
	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.
	Hakamatsuka, T. and Tanaka, N. 1997. Screening for bioactive compounds targeting the cellular signal transduction pathway using an RT‐PCR‐based bioassay system. Biol. Pharm. Bull. 20:464‐466.
	Heid, C.A., Stevens, J., Livak, K.J., and Williams, P.M. 1996. Real time quantitative PCR. Genome Res. 6:986‐994.
	Kitchen, R.R., Kubista, M., and Tichopad, A. 2010. Statistical aspects of quantitative real‐time PCR experiment design. Methods 50:231‐236.
	Maley, D., Mei, J., Lu, H., Johnson, D.L., and Ilyin, S.E. 2004. Multiplexed RT‐ PCR for high throughput screening applications. Comb. Chem. High Throughput Screen 7:727‐732.
	Phelan, M. 2007. Basic techniques in mammalian cell tissue culture. Curr. Protoc. Cell Biol. 36:1.1.1‐1.1.15.
	Rudnicki, S. and Johnston, S. 2009. Overview of liquid handling instrumentation for high‐throughput screening applications. Curr. Protoc.Chem. Biol. 1:43‐54.
	Schlesinger, J., Tönjes, M., Schueler, M., Zhang, Q., Dunkel, I., and Sperling, S.R. 2010. Evaluation of the LightCycler 1536 Instrument for high‐throughput quantitative real‐time PCR. Methods 50:S19‐S22.
	Schmittgen, T.D., and Livak, K.J. 2008. Analyzing real‐time PCR data by the comparative C(T) method. Nat. Protocols 3:1101‐1108.
	Swamidass, S.J., Bittker, J.A., Bodycombe, N.E., Ryder, S.P., and Clemons, P.A. 2011. An economic framework to prioritize confirmatory tests after a high‐throughput screen. J. Biomol. Screen. 15:680‐686.
	Swartzman, E., Shannon, M., Lieu, P., Chen, S.M., Mooney, C., Wei, E., Kuykendall, J., Tan, R., Settineri, T., Egry, L., and Ruff, D. 2010. Expanding applications of protein analysis using proximity ligation and qPCR. Methods 50:S23‐S26.
	Swinney, D.C. and Anthony, J. 2011. How were new medicines discovered? Nat. Rev. Drug Discov. 10:507‐519.
	Tuomi, J.M., Voorbraak, F., Jones, D.L., and Ruijter, J.M. 2010. Bias in the Cq value observed with hydrolysis probe based quantitative PCR can be corrected with the estimated PCR efficiency value. Methods 50:313‐322.
	VanGuilder, H.D., Vrana, K.E., and Freeman, W.M. 2008. Twenty‐five years of quantitative PCR for gene expression analysis. Biotechniques 44:619‐626.
	Wagner, B.K. and Arany, Z. 2009. High‐throughput real‐time PCR for detection of gene‐expression levels. In Cell‐Based Assays for High‐Throughput Screening (P.A. Clemons, N.J. Tolliday, and B.K. Wagner, eds.) pp. 167‐175. Humana Press, New York.
	Zhang, X.D. 2011. Illustration of SSMD, z score, SSMundefined, undefined score, and t statistic for hit selection in RNAi high‐throughput screens. J. Biomol. Screen. 16:775‐785.
	Zhang, J.H., Chung, T.D., and Oldenburg, K.R. 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4:67‐73.
Internet Resources
	http://www.biorad.com/genomics
	Bio‐Rad gene expression gateway. Provides useful tutorials on experimental design and detection formats.
	http://www.roche‐applied‐science.com/sis/realtimeready/index.jsp
	Roche Real‐Time Ready system. Search of existing qPCR detection reagents and bioinformatics tools for designing custom assays.
	https://www5.appliedbiosystems.com/tools/cadt/
	Life Technologies/Applied Biosystems, designer for custom Taqman probes and Taqman assays.

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

High‐Throughput RT‐PCR for Small‐Molecule Screening Assays

Abstract

Table of Contents

Materials

Basic Protocol 1: Two‐Step cDNA Generation and qPCR Analysis

Alternate Protocol 1: Generation of cDNA Using Oligo‐dT Capture Plates

Alternate Protocol 2: One‐Step qRT‐PCR

Figures

Videos

Literature Cited