RNA Expression: qRT-PCR
LifeTechnologies qRT-PCR (Taqman) Assays
Life Technologies offers more than 1,000,000 TaqMan Gene Expression Assays, the most comprehensive set of predesigned Real-Time PCR assays available. All TaqMan® Gene Expression Assays have been designed through a validated bioinformatics pipeline, and run with the same PCR conditions, eliminating the need to design primers or optimize PCR conditions. There are more than 1,200,000 TaqMan Gene Expression Assays available from 19 species.
TaqMan® Gene Expression Assays were used as the gold standard in the MicroArray Quality Control (MAQC) Project, which compared data from seven microarray platforms (Nature Biotechnology, September 2006).
TaqMan Gene Expression Assays have the highest specificity, greatest sensitivity, and largest dynamic range of any gene expression technology.
For more information regarding TaqMan Gene Expression Assays, or to design a project with the CBF, please contact Lori Kelly.
SABiosciences SYBR Green qRT PCR Assays
PCR Arrays are the most reliable tools for analyzing the expression of a focused panel of genes. Each 96-well plate, 384-well plate, or 100-well disc PCR Array includes SYBR Green-optimized primer assays for a thoroughly researched panel of relevant, pathway- or disease-focused genes. PCR Arrays can also be customized to contain a panel of genes tailored to your specific research interests. Our high-quality primer design and master mix formulation enable the PCR Array to amplify 96 or 384 different gene-specific products simultaneously under uniform cycling conditions. This combination provides the PCR Array with the specificity and the high amplification efficiencies required for accurate real-time SYBR Green results. The simplicity of the PCR Arrays makes them accessible for routine use in every research laboratory.
New developments in PCR Array technology enable optimal performance of RT2 Profiler PCR Arrays using RNA prepared from regular samples (0.1 - 5 ug RNA), FFPE samples, and small samples (1 - 100 ng RNA).
- Sensitivity: With the sensitivity of the RT2 First Strand Kit, as little as 1 ng or as much as 5 µg of total RNA per array plate provides greater than 80 percent present call rates.
- Reproducibility: The high reproducibility of the system, with replicate correlation coefficients > 0.99, means that experimental samples can be reliably compared across plates and runs.
- Specificity: The specificity of the system, imparted by the combination of SYBR Green primers and PCR master mixes, guarantees a single product of the predicted size from every reaction without secondary products such as primer dimers.
The complete PCR Array System guarantees the optimal performance of the PCR Array. Including the optional RT2 RNA QC PCR Array into the complete system maximizes control over consistent RNA quality.
Technology and Equipment
Life Technologies TaqMan Expression Assays
SABiosciences Expression Assays
Life technologies Prism 7900 HT
The Windows-based ABI Prism 7900 HT Systems use Peltier-based thermal cycling, TaqMan chemistry, and extended-life 488 nm argon-ion laser excitation to detect and quantitate nucleic acid sequences. interchangeable thermal cycling block formats let you select the format that is right for your project, using industry-standard 96- and 384-well formats, as well as a novel 384-well TaqMan® Low Density Array and a new Fast 96-well block that reduces run times from 2 hours to about 30 minutes. An easy automation upgrade path lets you add features to meet throughput demands. A Fast PCR option reduces run times from 2 hours to about 30 minutes.
This technology is very sensitive in that it is able to detect single copies of genes and requires very little starting material. The system monitors PCR at each cycle of a reaction, and estimates the cycle when the reaction reaches log phase increments (when the most useful quantitative information about the sample is available).
Quantitation can be “relative” to an internal standard such as a housekeeping gene, or “absolute” when compared to a standard curve generated from known concentrations. RT-PCR can be performed using a dual-labeled sequence-specific probe that carries both exciter and quencher tags. The exciter is cleaved from the quencher by the exonuclease activity of Taq polymerase and detected by the fluorometer. Another method of detection employs Sybr green detection. This method is less expensive and no sequence specific probe is required. Sybr green intercalates with double-stranded DNA and fluoresces as the PCR products are formed. This is detected and quantitated by the software.