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.

The TaqMan Assay
Currently five different companies, TaqMan® (Applied Biosystems, Foster City, CA, USA), TaqMan Style probes from IDT, 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.
Possible Applications
  • Gene Expression
  • SNP Detection
  • Single Nucleotide Polymorphism Detection
  • SNV Detection
  • Single Nucleotide Variants Detection
  • Plus/Minus (Presence/Absence) Assays
  • MicroRNA Profiling
  • MicroRNA Validation
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.
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. 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.
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.
Run Only
As an alternative, you can prepare a plate or card yourself and will only be charged for the equipment usage.
  • Applied Biosystems QuantStudio 7 Flex with TLDA card adapter

To order assays, please email qpcr@genome.med.harvard.edu. In the email, please include your user name and the assay number.

To order cards, please email qpcr@genome.med.harvard.edu. In the email, please include your user name and the type of card, including the species.

Gene Expression
  • Submit 500-1000ng of total RNA for each sample.
  • Each RT reaction can run up to ten assays so you only need submit for more than one RT if you have more than 10 assays.
MicroRNA
  • Submit 10ng of total RNA per assay being tested, plus one additional volume of 10ng.
  • If possible, submit in less than 3ul per 10ng.
  • Design your experiments including at least one normalizing assay that must be run on each RNA.
Plus/Minus
  • Contact the facility to discuss custom assays.
SNP/SNV
  • Submit your DNA with a concentration of at least 20ng/ul.
TLDA Cards
  • Contact the facility for specifics on custom cards.
  • MicroRNA cards require at least 500ng/card of total RNA.
Name Harvard Internal Harvard Affiliate/Academic Commercial
Gene Expression Plate Setup $233.64 $268.69 $303.74
Gene Expression Plate Well $15.18 $17.46 $19.74
Gene Expression TLDA Card Setup $169.70 $195.16 $220.61
Gene Expression TLDA Sample $9.46 $10.88 $12.30
Name Harvard Internal Harvard Affiliate/Academic Commercial
Micro RNA Plate Setup $164.22 $188.85 $213.49
Micro RNA Plate Well $16.23 $18.67 $21.10
Micro RNA TLDA Card Setup $193.81 $222.88 $251.95
Micro RNA TLDA Sample $9.29 $10.68 $12.08
Name Harvard Internal Harvard Affiliate/Academic Commercial
Plus / Minus Plate Additional Control $1.27 $1.46 $1.65
Plus / Minus Plate Setup $109.50 $125.93 $142.35
Plus / Minus Plate Well $3.02 $3.47 $3.93
Name Harvard Internal Harvard Affiliate/Academic Commercial
SNP Plate Setup $114.68 $131.88 $149.09
SNP Plate Well $1.31 $1.50 $1.70
SNP TLDA Card Setup $169.70 $195.16 $220.61
SNP TLDA Sample $9.46 $10.88 $12.30
Name Harvard Internal Harvard Affiliate/Academic Commercial
Run Only Plate Fee $150.00 $172.50 $195.00
Run Only TLDA Card Fee $150.00 $172.50 $195.00

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 (Absolute Quantification)
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 (Relative Quantification)
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-δδCt method, where
δδ Ct = δCt, sample - δCt, reference
Here, δ Ct sample is the Ct value for any sample normalized to the endogenous housekeeping gene and δ Ct reference is the Ct value for the calibrator also normalized to the endogenous housekeeping gene.
For the δδ 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 δ 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.
Data Results
Our software can do either method.
  • For the AQ method, you will receive a text file with the sample name/location and a Ct value.
  • For the RQ method you will receive a text file with the following
    Ct values
    Delta Ct (sample Ct- endogenous control Ct from the same sample)
    Delta delta Ct (sample delta Ct - calibrator sample delta Ct)
    Relative Quantification (ratio of delta delta Ct values where the calibrator value is 1 )