From GenoSeq

TAQMAN ABI7900

Download the User Bulletin #2, #5, TaqMan Human Endogenous Control Plate Protocol, and Primer/Probe Optimization Protocol from Applied Biosystems website Support - Documents on Demand - Applied Biosystems.

If using Taqman Fluorescent probes, send us the sequence of your gene of interest (The length of sequence should be between 150-250bp.) via email to uma@ucla.edu OR webseq@genetics.ucla.edu as a Word document attachment. Once we have emailed you the sequences for Primers/Probes sequences, order them from the vendor of your choice.

• Applied Biosystems
• Qiagen
• Superarray
• Biosearch
• Idaho Technologies, Inc.
• Synthegen, LLC
• Genset Corp.
• Integrated DNA Technologies
• Gene Express National Enterprises

If you do not need the probes and are using the SYBR green master mix or SYBR green reagents then please make sure to make your primers using any of the following programs or your choice of primer software. Do not eyeball the primers.

• Primer3 (Whitehead Institute)
• Web Primers (University of Minnesota)
• Raw Primer (University of Minnesota)
• Web Primer (Stanford University)
• Gene Primer (University of Southern California)

A frequently updated list of validated primer/probe sets for quantitation of a variety of mRNAs is maintained at Real Time Primers

At this time also order the consumables like optical plates/tubes, covers/caps, compression pads, master mix from any of the following vendors or vendors of your choice.

• Island Scientific
• Applied Biosystems
• Denville Scientific
• ISC BioExpress
• BD Biosciences

Make an appointment with The UCLA Sequencing Core for Taqman run time slot by logging on to our website.

• Webseq. Our Phone # is 310-825-5488. We are located in 5309 Gonda Bldg.

When you set up your samples, please print and use the 96 well format provided here to mark your samples, dyes used, volume and PCR conditions.

Optimize your primers/probe concentrations as described in user bulletin #2.

This will be your first run.

Calculate the dilution factor for your primers and probes: Calculate Primer Dilution

Calculate the DNA dilution use this website: Calculate DNA Dilution

After looking at your data, you will see which concentration is optimum for your samples.

Interpretation of results

At the end of each reaction, the recorded fluorescence intensity is used for the following calculations:

Rn⁺ is the Rn value of a reaction containing all components, Rn^- is the Rn value of an unreacted sample (baseline value or the value detected in NTC). ΔRn is the difference between Rn⁺ and Rn^-. It is an indicator of the magnitude of the signal generated by the PCR.

There are three methods to quantitate the amount of template:

1. Absolute standard method: In this method, a known amount of standard such as in vitro translated RNA (cRNA) is used,

2. Relative standard: Known amounts of the target nucleic acid are included in the assay design in each run,

3. Comparative C^T method: This method uses no known amount of standard but compares the relative amount of the target sequence to any of the reference values chosen and the result is given as relative to the reference value (such as the expression level of resting lymphocytes or a standard cell line).

The comparative C^T method (ΔΔC^T) for relative quantitation of gene expression

This method enables relative quantitation of template and increases sample throughput by eliminating the need for standard curves when looking at expression levels relative to an active reference control (normalizer). For this method to be successful, the dynamic range of both the target and reference should be similar. A sensitive method to control this is to look at how ΔC^T (the difference between the two C^T values of two PCRs for the same initial template amount) varies with template dilution. If the efficiencies of the two amplicons are approximately equal, the plot of log input amount versus ΔC^T will have a nearly horizontal line (a slope of <0.10). This means that both PCRs perform equally efficiently across the range of initial template amounts. If the plot shows unequal efficiency, the standard curve method should be used for quantitation of gene expression. The dynamic range should be determined for both (1) minimum and maximum concentrations of the targets for which the results are accurate and (2) minimum and maximum ratios of two gene quantities for which the results are accurate. In conventional competitive RT-PCR, the dynamic range is limited to a target-to-competitor ratio of about 10:1 to 1:10 (the best accuracy is obtained for 1:1 ratio). The real-time PCR is able to achieve a much wider dynamic range.

Running the target and endogenous control amplifications in separate tubes and using the standard curve method requires the least amount of optimization and validation. The advantage of using the comparative C^T method is that the need for a standard curve is eliminated (more wells are available for samples). It also eliminates the adverse effect of any dilution errors made in creating the standard curve samples.

As long as the target and normalizer have similar dynamic ranges, the comparative C^T method (ΔΔC^T method) is the most practical method. It is expected that the normalizer will have a higher expression level than the target (thus, a smaller C^T value). The calculations for the quantitation start with getting the difference (ΔC^T) between the C^T values of the target and the normalizer:

ΔC^T = C^T (target) - C^T (normalizer)

This value is calculated for each sample to be quantitated (unless, the target is expressed at a higher level than the normalizer, this should be a positive value. It is no harm if it is negative). One of these samples should be chosen as the reference (baseline) for each comparison to be made. The comparative ΔΔC^T calculation involves finding the difference between each sample's ΔC^T and the baseline's ΔC^T. If the baseline value is representing the minimum level of expression, the ΔΔC^T values are expected to be negative (because the ΔC^T for the baseline sample will be the largest as it will have the greatest ΔC^T value). If the expression is increased in some samples and decreased in others, the ΔΔC^T values will be a mixture of negative and positive ones. The last step in quantitation is to transform these values to absolute values. The formula for this is:

comparative expression level = 2 - ΔΔC^T

For expressions increased compared to the baseline level this will be something like 23 = 8 times increase, and for decreased expression it will be something like 2-3 = 1/8 of the reference level. Microsoft Excel can be used to do these calculations by simply entering the C^T values (there is an online ABI tutorial on the use of spread sheet programs to produce amplification plots; the TaqMan Human Endogenous Control Plate TaqMan Human Endogenous Control Plate protocol also contains detailed instructions on using MS Excel for real-time PCR data analysis).