Gene Expression Quantification

Since the inception of the Genomics and Sequencing Core in 1998, we have processed thousands of real-time quantitative PCR (qPCR) plates, each containing approximately 96 reactions/plate. We have extensive knowledge of all aspects of RT-qPCR, and more recently, digital PCR. We can assist the customer in experimental design, primer/probe design, absolute and relative quantitation, normalization, and analysis. Currently, we use two methodologies to evaluate gene expression levels:

I. Quantitative Real-Time PCR. Genomics and Sequencing Core offers full service (including labor, reagents, and plastics for a plate setup of two genes, qPCR run, and final data analysis) or offers instrument use on the charge per run basis.

TaqMan: Applied Biosystems, Inc. (ABI, now ThermoFisher Scientific, Inc.) has developed the TaqMan method to overcome the limitations of standard end-point PCR. Through the use of specific target probes incorporating MGB (minor groove binder) technology, amplification of genes is monitored in real-time by fluorescent emission that is proportional to the number of amplicons in the sample. Pre-designed TaqMan gene expression assays are available from ABI. These are kits that include pre-designed and validated primers and probes for more than 30 species including human, mouse, and rat, Arabidopsis, Drosophila and C. elegans. There are over 2.3 million probe and primer sets to choose from.

The TaqMan® Gene Expression Array Plates are 96-well plates, designed for use with a variety Real-Time PCR Systems, including 7500 Real-Time PCR System. Ideal for screening biomarkers and toxicology panels, and for analyzing pathways, target classes, and complete disease sets using real-time PCR.

The TaqMan® Gene Expression Array Cards are 384-well micro fluidic cards (arrays), designed for use with the Applied Biosystems QuantStudio 7 and 7900HT Fast Real-Time PCR Systems, among others, to perform hundreds of real-time PCR reactions simultaneously. Good for simultaneous validation of tens to hundreds hits from microarray or RNA-Seq experiments. Simple to implement and available in a number of formats, TaqMan Arrays require minimal amounts of sample, and allow for 1 to 8 samples to be run in parallel against 12 to 384 TaqMan® Gene Expression Assay targets.

SYBR Green: SYBR Green dye chemistry works by binding to double stranded DNA products formed during the PCR reaction. It is a relatively inexpensive method but has its limitations such as generating false positive signals as a result of it binding to non-specific DNA products. The Genomics and Sequencing Core is happy to provide the SYBR Green option for screening your samples for expression of a target.

MicroRNA detection: MicroRNAs (miRNA) are non-coding RNAs that are capable of silencing translation of many mRNA targets. They are small (~22 nucleotides) and use the RNA interference pathway to exert their effects. Applied Biosystems, Inc. has developed a way of detecting these small biologically active, mature miRNAs using TaqMan technology. Probes specific for these sequences detect and amplify in a RT-qPCR-based reaction to allow the investigator to quantify levels of these transcripts in their samples. A very popular product is the TaqMan Array MicroRNA 384-well Cards  which simultaneously interrogate 384 TaqMan microRNA assays in parallel. These plates can be run at the Genomics and Sequencing Core in the ABI QuantStudio 7 or 7900HT Fast Real-Time PCR System.

II. Digital PCR. Genomics and Sequencing Core offers full service (including labor, reagents, and plastics for a plate setup, droplet generation, PCR, droplets reading, and final analysis) or offers instrument use on the charge per run basis.

The Genomics and Sequencing Core has acquired the QX200 Droplet Digital PCR System from Bio-Rad  Often referred to as third generation PCR technology, the QX200 offers direct absolute quantitation of targets, whether cDNA, genomic DNA, or pathogen RNA or DNA using standard TaqMan probes and primers. It is especially well suited for rare event detection, such as that occurring in HIV latency. We currently offer ddPCR assays for HIV DNA (2-LTR circles, Gag, Pol) and RNA (Gag, Tat-Rev, total polyadenylated HIV RNA). We also run ddPCR with any TaqMan assays of interest.

Pricing*

*Pricing is for UCSD/VMRF/VA customers only. All others should contact the Core for pricing.