Quantitative PCR:

The Biopolymers Facility offers a variety of Quantitative PCR assays using the Applied Biosystems 7900 HT Fast instrument and ABI TaqMan assays.

If you have any questions, please contact our qPCR core technician at: qpcr@genome.med.harvard.edu

General Technology Overview

Services/Pricing

General Technology Overview

(the following excerpts are taken from the ABI / Ambion websites)

RT-PCR (reverse transcription-polymerase chain reaction) is the most sensitive technique for mRNA detection and quantitation currently available. Compared to the two other commonly used techniques for quantifying mRNA levels, Northern blot analysis and RNase protection assay, RT-PCR can be used to quantify mRNA levels from much smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell. Furthermore, real-time RT-PCR has become the preferred method for validating results obtained from array analyses and other techniques that evaluate gene expression changes on a global scale.

PCR Review

The TaqMan Assay

Data Analysis / Quantitation of Results

PCR Review:

At the start of a PCR reaction, reagents are in excess, template and product are at low enough concentrations that product renaturation does not compete with primer binding, and amplification proceeds at a constant, exponential rate. The point at which the reaction rate ceases to be exponential and enters a linear phase of amplification is extremely variable, even among replicate samples, but it appears to be primarily due to product renaturation competing with primer binding (since adding more reagents or enzyme has little effect). At some later cycle, the amplification rate drops to near zero (plateaus), and little more product is made.

For the sake of accuracy and precision, it is necessary to collect quantitative data at a point in which every sample is in the exponential phase of amplification (since it is only in this phase that amplification is extremely reproducible). Analysis of reactions during exponential phase at a given cycle number should theoretically provide several orders of magnitude of dynamic range. Rare targets will probably be below the limit of detection, while abundant targets will be past the exponential phase. In practice, a dynamic range of 2-3 logs can be quantitated during end-point relative RT-PCR. In order to extend this range, replicate reactions may be performed for a greater or lesser number of cycles, so that all of the samples can be analyzed in the exponential phase. Real-time PCR automates this otherwise laborious process by quantitating reaction products for each sample in every cycle. The result is an amazingly broad 107-fold dynamic range, with no user intervention or replicates required. Data analysis, including standard curve generation and copy number calculation, is performed automatically.

The TaqMan Assay:

Currently four different chemistries, TaqMan® (Applied Biosystems, Foster City, CA, USA), Molecular Beacons, Scorpions® and SYBR® Green (Molecular Probes), are available for real-time PCR.

TaqMan probes depend on the 5'- nuclease activity of the DNA polymerase used for PCR to hydrolyze an oligonucleotide that is hybridized to the target amplicon. TaqMan probes are oligonucleotides that have a fluorescent reporter dye attached to the 5' end and a quencher moeity coupled to the 3' end. These probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the proximity of the fluorophore and the quench molecules prevents the detection of fluorescent signal from the probe. During PCR, when the polymerase replicates a template on which a TaqMan probe is bound, the 5'- nuclease activity of the polymerase cleaves the probe. This decouples the fluorescent and quenching dyes and frees the fluorescent probe to be detected. Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavage. Well-designed TaqMan probes require very little optimization. In addition, they can be used for multiplex assays by designing each probe with a spectrally unique fluor/quench pair. However, TaqMan probes can be expensive to synthesize, with a separate probe needed for each mRNA target being analyzed.

TaqMan probes, Molecular Beacons and Scorpions allow multiple DNA species to be measured in the same sample (multiplex PCR), since fluorescent dyes with different emission spectra may be attached to the different probes. Multiplex PCR allows internal controls to be co-amplified and permits allele discrimination in single-tube, homogeneous assays. These hybridization probes afford a level of discrimination impossible to obtain with SYBR Green, since they will only hybridize to true targets in a PCR and not to primer-dimers or other spurious products.

Quantitation of Results:

Two strategies are commonly employed to quantify the results obtained by real-time RT-PCR; the standard curve method and the comparative threshold method. These are discussed briefly below.

Standard Curve Method:

In this method, a standard curve is first constructed from an RNA of known concentration. This curve is then used as a reference standard for extrapolating quantitative information for mRNA targets of unknown concentrations. Though RNA standards can be used, their stability can be a source of variability in the final analyses. In addition, using RNA standards would involve the construction of cDNA plasmids that have to be in vitro transcribed into the RNA standards and accurately quantitated, a time-consuming process. However, the use of absolutely quantitated RNA standards will help generate absolute copy number data. In addition to RNA, other nucleic acid samples can be used to construct the standard curve, including purified plasmid dsDNA, in vitro generated ssDNA or any cDNA sample expressing the target gene. Spectrophotometric measurements at 260 nm can be used to assess the concentration of these DNAs, which can then be converted to a copy number value based on the molecular weight of the sample used. cDNA plasmids are the preferred standards for standard curve quantitation. However, since cDNA plasmids will not control for variations in the efficiency of the reverse transcription step, this method will only yield information on relative changes in mRNA expression. This, and variation introduced due to variable RNA inputs, can be corrected by normalization to a housekeeping gene.

Comparative Ct Method:

Another quantitation approach is termed the comparative Ct method. This involves comparing the Ct values of the samples of interest with a control or calibrator such as a non-treated sample or RNA from normal tissue. The Ct values of both the calibrator and the samples of interest are normalized to an appropriate endogenous housekeeping gene.

The comparative Ct method is also known as the 2-[delta][delta]Ct method, where

[delta][delta] Ct = [delta]Ct, sample - [delta]Ct, reference

Here, [delta] Ct sample is the Ct value for any sample normalized to the endogenous housekeeping gene and [delta] Ct reference is the Ct value for the calibrator also normalized to the endogenous housekeeping gene.

For the [delta][delta] Ct calculation to be valid, the amplification efficiencies of the target and the endogenous reference must be approximately equal. This can be established by looking at how [delta] Ct varies with template dilution. If the plot of cDNA dilution versus delta Ct is close to zero, it implies that the efficiency of each of the target and housekeeping genes are very similar. If a housekeeping gene cannot be found whose amplification efficiency is similar to the target, then the standard curve method is preferred.

In spite of the rapid advances made in the area of real-time PCR detection chemistries and instrumentation, end-point RT-PCR still remains a very commonly used technique for measuring changes in gene-expression in small sample numbers. End-point RT-PCR can be used to measure changes in expression levels using three different methods: relative, competitive and comparative. The most commonly used procedures for quantitating end-point RT-PCR results rely on detecting a fluorescent dye such as ethidium bromide, or quantitation of P32-labeled PCR product by a phosphorimager or, to a lesser extent, by scintillation counting.

Relative quantitation compares transcript abundance across multiple samples, using a co-amplified internal control for sample normalization. Results are expressed as ratios of the gene-specific signal to the internal control signal. This yields a corrected relative value for the gene-specific product in each sample. These values may be compared between samples for an estimate of the relative expression of target RNA in the samples; for example, 2.5-fold more IL-12 in sample 2 than in sample 1. Absolute quantitation, using competitive RT-PCR, measures the absolute amount (e.g., 5.3 x 105 copies) of a specific mRNA sequence in a sample. Dilutions of a synthetic RNA (identical in sequence, but slightly shorter than the endogenous target) are added to sample RNA replicates and are co-amplified with the endogenous target. The PCR product from the endogenous transcript is then compared to the concentration curve created by the synthetic "competitor RNA."

Comparative RT-PCR mimics competitive RT-PCR in that target message from each RNA sample competes for amplification reagents within a single reaction, making the technique reliably quantitative. Because the cDNA from both samples have the same PCR primer binding site, one sample acts as a competitor for the other, making it unnecessary to synthesize a competitor RNA sequence.

Both relative and competitive RT-PCR quantitation techniques require pilot experiments. In the case of relative RT-PCR, pilot experiments include selection of a quantitation method and determination of the exponential range of amplification for eachmRNA under study. For competitive RT-PCR, a synthetic RNA competitor transcript must be synthesized and used in pilot experiments to determine the appropriate range for the standard curve. Comparative RT-PCR yields similar sensitivity as relative and competitive RT-PCR, but requires significantly less optimization and does not require synthesis of a competitor.

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Services and Pricing

Gene Expression

The facility offers gene expression analysis using TaqMan assays from Applied Biosystems. Select from over 700,000 pre-designed and optimized assays for human, mouse, rat, Arabidopsis, Drosophila, C. elegans and Rhesus Macaque genes.

Available Endogenous Controls:

There has been significant debate over what constitutes a valid endogenous control in QPCR. If prior expression data exists from Affymetrix, Illumina or other whole genome expression studies, you can use this information to help determine a valid endogenous control. If you have no previous expression data, then select a housekeeping gene from the list below that seems most likely to be consistent across your samples.

Human:
Beta Actin
Beta-2-microglobulin
GAPDH
Beta-glucoronidase
HPRT1
PGK1
PPIA
RPLO
TBP
TFRC

Mouse:
Beta Actin
GAPDH

Rat:
Beta Actin
GAPDH

microRNAs

A simple, two-step protocol requires only reverse transcription with a miRNA-specific primer, followed by real-time PCR with TaqMan® probes. The assays target only mature microRNAs, not their precursors, ensuring biologically relevant results.

Plus/Minus Assays

Please contact the facility to discuss custom assays.

SNPs:

Order custom, single tube TaqMan® reagent-based assays to perform genotyping studies with any possible SNP in any organism. Three different size assays can be ordered to meet your project needs. Custom TaqMan® SNP Genotyping Assays support both SNPs and insertion-deletions of up to six bases, and multiple Nucleotide Polymorphisms.

* Assay is provided by customer *

SNP assays are available from Applied Biosystems in four categories:

TaqMan® Validated & Coding SNP Genotyping Assays:

This assay set contains 160,000 small scale, validated SNP assays with 20 million associated genotypes that are held in inventory. In addition there are 30,000 small scale, coding SNP assays held in inventory.

TaqMan® Pre-Designed SNP Genotyping Assays:

This assay set contains 3.5 million Pre-Designed SNP assays, including 2.7 million HapMap and 30,000 coding SNP assays available in small, medium and large scale. These assays are made-to-order.

TaqMan® Pre-Developed Assay Reagents for Allelic Discrimination:

These assays use the 5' nuclease assay to genotype purified DNA samples for specific mutations. Most TaqMan® PDARs for AD assays discriminate between two alleles of single nucleotide polymorphisms (SNPs). Each assay contains two different TaqMan® probes, and each uniquely labeled probe binds preferentially to one of the alleles.

TaqMan® Pre-Developed Assay Reagents for Drug Metabolism:

Detect biologically important polymorphisms that reside in complex portions of the genome. These polymorphisms have been associated with certain diseases such as cancer, and have been shown to significantly impact drug efficiency. Where possible all assays have been mapped to the common public allele nomenclature. All TaqMan® DME genotyping assays have proven performance across 180 unique DNA samples. Genotype over 2,400 high value polymorphisms located in regulatory elements and coding regions for 220 drug metabolism and transporter genes.

TLDA Card

TLDA or TaqMan Low Density Array cards are 384 well plates preloaded with the TaqMan assays of your choice along with endogenous controls.

Due to the varied nature of custom TLDA cards, we have split our pricing into the various components of the procedure, so you can select only the steps you need.

* While we do not require this step, due to the high cost of the reagents and cards, we highly recommend it

Run-Only Experiments

As an alternative, you can prepare a plate or card yourself and will only be charged for the equipment usage.

Select Downloads:

TaqMan Assay Universal Mix Protocols

TaqMan Gene Expression Product Brochure

TaqMan Chemistry Guide

TaqMan microRNA Product Brochure

microRNA TaqMan Article

microRNA TaqMan Assay Protocol

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©2004 Harvard Medical School