Guide to Performing Relative Quantitation of Gene Expression Using Real-Time
Quantitative PCR
Guide to Performing Relative Quantitation of Gene Expression
Using Real-Time Quantitative PCR
Table of Contents
Section I: Introduction to Real-Time PCR and Relative Quantitation of Gene Expression
1. Introduction
2. What is Relative Quantitation?
3. Terms and Acronyms
4. Relative Quantitation of Gene Expression Requires the Quantitation of Two
Different Genes (Target and Endogenous Control)
5. Factors Affecting Accurate Real-Time PCR Results
6. What is PCR Amplification Efficiency?
Section II: RNA Preparation and Rever Transcription
1. Introduction
2. Quantifying Input RNA
3. Rever Transcription (RT) for Relative Quantitation of Gene Expression
4. Selecting Rever Transcription and Real-Time PCR Reagents
5. Determination of Input RNA Amounts for a Relative Quantitation
Study
6. Identifying PCR Inhibition
7. How Much Genomic DNA Contamination can be Tolerated in a Relative
Quantitation of Gene Expression Assay?
Section III: Assay Selection and Design for Relative Quantitation
Selecting or Designing Primers and TaqMan® Probes for Relative Quantitation of
Gene Expression
1. TaqMan® Gene Expression Assays
2. Custom TaqMan® Gene Expression Assays
3. TaqMan® Pre-Developed Assay Reagents (TaqMan® PDARs)
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4. U of Primer Express® Software for the Design of Primer and Probe Sets for
Relative Quantitation of Gene Expression
5. Design of Assays for SYBR® Green I Applications
Section IV. Identification and Selection of Endogenous Controls for Relative Quantitation
1. Uniformity of Endogenous Control Expression.
2. Validation of Target and Control Genes for the Comparative C T Method
3. Multiplexing Endogenous Controls and Target Genes
Section V. Customized and Pre-Configured Relative Quantitation Gene Expression Products
1. TaqMan® Low Density Arrays (7900HT Microfluidic Cards)
2. Pre-Configured TaqMan® Low Density Arrays (Immune Profiling)
成本核算的方法有哪些3. TaqMan® Cytokine Gene Expression Plate
4. TaqMan® Human Endogenous Control Plate
Section VI. Ordering Real Time PCR Reagents
Section VII. Relative Quantitation of Gene Expression Experimental Design and Analysis
1. Introduction
2. The Relative Standard Curve Method日剧smile
a. Example of the Standard Curve Method: Using an Independent Sample
for a Standard Curve
b. Standard Deviation Calculations Using the Standard Curve Method
3. The Comparative Ct Method (∆∆C T Method)
a. A Validation Experiment is Necessary to Determine if your ∆∆C T
Calculation is Valid
ableb. Plotting the Results of the Validation Experiment
c. Validation Experiment Results
d. The Comparative C T Method (∆∆C T Method): Data Analysis Example
e. What if a ∆∆C T Value is Positive?
Appendix A Definitions
Appendix B Reagents, Protocols, and Supporting Documentation
Section I
Introduction to Real-Time PCR and
Relative Quantitation of Gene Expression
1. Introduction谢谢你们英语怎么说
Real-time quantitative PCR offers rearchers a powerful tool for the quantitation of target nucleic acids. To understand the value that real-time PCR provides over traditional PCR methods and to obtain information on chemistries and strategies, you can review:
Real Time PCR vs. Traditional PCR
Esntials of Real Time PCR
This tutorial guides you through performing relative quantitation of gene expression using real-time P
CR technologies developed by Applied Biosystems. It assists you in understanding the foundations of relative quantitation and provides guidance for lecting assays, experimental strategies, and methods of data analysis. The information prented is relevant for instrumentation, reagents, and consumables provided by Applied Biosystems. This tutorial expands on many of the topics that are introduced in Ur Bulletin #2: Relative Quantitation of Gene Expression. Throughout this tutorial there are many hyperlinks to external sites, documentation, and links to pages within this document. After you go to one of the hyperlinks, click the back button on your browr to return to your original location in the document.
Applied Biosystems offers a variety of systems on which real-time quantitative PCR can be performed. The real-time PCR instruments are:
• Applied Biosystems 7300 Real-Time PCR System
cannot• Applied Biosystems 7500 Real-Time PCR System
• Applied Biosystems 7900HT F ast Real-Time PCR System
新东方考研怎么样• ABI® PRISM 7000 Sequence Detection System
2. What is Relative Quantitation?
Methods for relative quantitation of gene expression allow you to quantify differences in the expression level of a specific target (gene) between different samples. The data output is expresd as a fold-change or a fold-difference of expression levels. For
example you might want to look at the change in expression of a particular gene over a given time period in treated vs. untreated samples. For this hypothetical study, you can choo a calibrator sample (i.e. untreated at day 0) and an endogenous control gene to normalize input amounts. For all samples, levels of both target and endogenous control genes would be assd by real-time PCR. The results (target levels normalized to endogenous control levels) would then be expresd in a format such as “At day 30, sample A had a 10-fold greater expression level of the target gene than at day 0”.
If you want to obtain absolute quantities of gene targets you need to perform absolute quantitation, which is beyond the scope of this document.
3. Terms and Acronyms – The following terms and acronyms are ud throughout this
document. Additional information on specific definitions is available in the Appendix or by clicking
the appropriate links.
Terms/Acronyms Definition Active reference An active signal ud to normalize experimental results. Endogenous controls are an
英国大学排名2013example of an active reference. Active reference means the signal is generated as a
result of PCR amplification. The active reference has its own t of primers and probe. Amplicon A PCR product generated from a DNA or cDNA template.incity
Amplification efficiency The rate at which a PCR amplicon is generated, commonly measured as a percentage value. If a particular PCR amplicon doubles in quantity during the geometric pha of its PCR amplification then the PCR assay is said to have 100% efficiency. The value assigned to the efficiency of a PCR reaction is a measure of the overall performance of a real-time PCR assay.
Baline The background fluorescence signal emitted during the early cycles of the PCR reaction be
fore the real-time PCR instrument detects the amplification of the PCR product. Calibrator A sample ud as the basis for comparative expression results
C T Threshold cycle. The C T is the cycle number at which the fluorescence generated within
a reaction cross the threshold line. C T values are logarithmic and are ud either
directly (comparative C T method) or indirectly (interpolation to standard curves to create
linear values) for quantitative analys.
Custom TaqMan®Gene Expression Products1Custom TaqMan® Gene Expression Assays are products designed, synthesized, and delivered as pre-mixed primers and TaqMan® MGB probe ts bad on quence information submitted by the customer.
Custom TaqMan®Genotyping Products2Custom TaqMan® Genotyping Assays are products designed, synthesized, and delivered as a t of pre-mixed primers and TaqMan® MGB probes bad on quence information submitted by the customer.
Dynamic range The range (maximum to minimum) of sample concentrations or input amounts that a given assay is capable of detecting.
Endogenous control A gene quence contained in a sample that is ud to normalize target quantities. In addition to the target quence, an endogenous control is quantified as a means of correcting results that can be skewed by input nucleic acid loading differences. Endogenous controls are an example of an active reference.
Experimental replicate An amplification that us the same PCR reagents as another amplification and that us template preparations from similar but not identical samples. Experimental replicates provide information about the overall precision of the experiment. For example, if you want to examine the effect of drug treatment on the level of a mou mRNA, you would treat multiple mice identically with the drug to determine the variation of respon in the mou population. A group of ten mice would reprent ten experimental replicates.
Identical replicate An amplification performed in multiple wells using the same template preparation and the same PCR reagents. Identical replicates provide:
• Data prervation: If amplification fails in one well, replicates in other wells can
potentially provide data.
• Monitoring: Replicates can be ud to monitor the precision of the PCR amplification and detection steps.
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Passive reference A dye that provides an internal fluorescence reference to which the reporter dye signal can be normalized during data analysis. The reference dye does not participate in the
PCR reaction. This normalization corrects for fluorescence fluctuations that are caud
by changes in reaction concentration or volume. Failure to u a passive reference dye
can compromi accurate target quantitation. Applied Biosystems incorporates the
internal passive reference dye ROX TM in all of its real-time PCR chemistries.
