Research at Fox Chase »Research Facilities »Biotechnology

• Real-time PCR
• DNA Synthesis
• Mass Spectrometry
• Proteomic services
• HPLC service

Function

BBF performs qRT-PCR reactions with suitable controls and normalization reference genes, high accuracy robotic pipetting, and data analysis, are all completed in a timely and economical manner. BBF provided services include: the validation of DNA array gene expression data, quantitation of gene expression of low abundance messages, determination of the zygosity of transgenic mice, quantitative analysis of alternative spliced transcripts, quantification of DNA in immunoprecipitated histone fractions (ChIP assays), and validation of the effectiveness of siRNA transcription silencing. SNP genotyping is not a focus of this Real-time PCR service except to provide consultation when requested. As an additional benefit, all primers and probes accumulated are kept in a database ready for sharing by all the users. We have developed about 200 assays and purchased about 200 pre-developed assays so far including many housekeeping genes and numerous cancer-related genes, in man and mouse.

Description

The BBF has maintained and provided support for a 48 cell Cepheid SMART Cycler for Real-time PCR applications since 2001. In 2002, we added an Applied BioSystems 7900HT Real-time PCR workstation, a Corbett Robotics PCR pipetting robot, an Agilent Bioanalyzer lab-on-a-chip system for determining the quality and quantity of RNA in samples, and a Transgenomic WAVE DHPLC system to determine the stability of the probes and the quality of the PCR products. Our instrument, the ABI 7900, supports the use of Sybr Green assays, Taqman assays, and FRET (Fluorescence Resonance Energy Transfer) assays. A Smartcycler has 32 independently programmable PCR wells is also available for user operation. Taqman reactions use probes of about 21 nucleotides long with a 5' fluorescent reporter molecule such as 6-FAM or TET, and a 3' quencher such as TEMPRA or one of the BHQs. When free in solution, the quencher molecule darkens the fluorescence of the reporter molecule (this is one form of FRET). The exonuclease activity of the DNA polymerase excises the reporter when the probe is hybridized to the PCR product, thereby becoming fluorescent. At Fox Chase, we can synthesize the BHQ version of Taqman probes. Combining the inherent stability of BHQ probes during DNA synthesis with the high quality of our DNA synthesis has led to BHQ FRET probes that can be used in Taqman reactions with minimal purification. About half of the 500 assays validated in this facility are prevalidated commercial assays. Experiments for which the assays are on-hand are often completed within 48 hours.