The Penn Molecular Profiling Facility provides instrumentation and expertise for DNA and RNA profiling. Microarrays and other highly parallel technologies provide the means for measuring the identity and abundance of DNA and RNA for targeted genes, or the whole genome, in a biological sample. The Facility offers a range of cost and performance options suitable for a variety of experimental questions. Molecular assays are critical to many aspects of basic, clinical, and population research, including molecular stratification of patients entering clinical protocols, molecular epidemiological and pharmacogenetic studies, as well as longitudinal follow-up of patients in clinical investigations.
Since molecular technologies and instrumentation are evolving rapidly, the centralization of molecular testing services within this core facilitates utilization of leading-edge molecular analyses by the investigators. Because some assays are used for clinical decisions during clinical trials, tests are meticulously designed and performed with strict attention to the prevention of polymerase chain reaction (PCR) contamination.
The Facility is a fully equipped molecular biology laboratory staffed by experienced individuals in developing and performing molecular biological assays. While the Facility staff performs most of the assays, investigator-performed studies are actively encouraged through the sharing of Facility procedures, individualized training of investigators or their technical staff, and use of core equipment.
The Facility Director and the Technical Director are available to talk with investigators to explore how the services of the facility can enhance or design their specific research projects. We invite investigators to meet with us in the planning stages of their studies, especially before grant submissions, to discuss services that the core can provide, such as budget information and a description of the core for the resources section of the grant, as well as to plan collection and handling of the specimens for the study. The Facility is happy to custom design assays to fit an investigator's needs.
"Gene expression profiling or microarray analysis has enabled the measurement of thousands of genes in a single RNA sample. GeneChip microarrays are manufacture by photochemical synthesis. Because of this manufacturing technology, more than a million different probes can be synthesized on an array roughly the size of a thumbnail. These numbers allow the inclusion of multiple probes to interrogate the same target sequence, providing statistical rigor to data interpretation. Over the years the GeneChip platform has proven to be a reliable and robust system, enabling many new discoveries and breakthroughs to be made by the scientific community.
After hybridization of the biotinylated sample, the chip is stained with streptavidin-phycoerythrin and read with a confocal scanner. Control and experimental samples are hybridized to separate chips. Comparison of the two chips is performed to determine the differential gene expression levels of the two compared samples."
"The Gene Titan family of instruments and proven GeneChip Array Plates from Affymetrix are the first to provide a hands-free, automated solution for monitoring gene expression.
The Gene Titan Multi-Channel (MC) Instrument automate array processing from target hybridization to data generation by combining a hybridization oven, fluidics processing, and state-of-the art imaging device into a single bench-top instrument.
The imaging device in the Gene Titan MC Instrument uses an external, high-intensity xenon lamp and dual excitation and emission filters to capture images from array plates for use in genome-wide expression and genotyping studies.
Key Features and Benefits of the Assay
• Flexible - Supports gene expression studies on 16-, 24- and 96-format array plates
• Accurate - Delivers high-quality, consistent data every time
• Efficient - Condenses hands-on processing time to as little as 30 minutes, images an array in less than five minutes, and operates unattended overnight
• Robust - Has fewer moving parts so it's easily maintained and rarely requires IT support
• Scalable - Meets both medium- and high-throughput needs
• Adaptable - Creates flexible workflows and sample registration via Affymetrix GeneChip Command Console (AGCC) Software"
Used to set up Taqman reactions in 384-well plates
"In the Chemagic system, magnetic separation is achieved through the use of an electromagnet and separation heads with rods that can be magnetized. The metal rods are immersed into the magnetic bead suspension. When the electromagnet is switched on, the rods become magnetic and the beads are separated. To resuspend separated magnetic bead pellet, the magnet is switched off and a stirring motor to which all rods are connected is switched on. This leads to a very effective and gentle resuspension process resulting in separation products with both highest yields and purities."
"This is the first high-throughput, target-enrichment system designed to work with all of the major next-generation sequencing instruments. The Access Array System enables the user to enrich multiple unique targets (such as exons) from a large number of samples, all at one time. The system combines the cost and throughput benefits of microfluidics with the proven performance and flexibility of PCR. Custom designed primer sets amplify (and tag) PCR products for sequencing.
Key Features and Benefits of the Assay
• Target Enrichment
• Sample Barcoding for Multiplexed Sequencing
• Sequencing Library Prep Using Amplicon Tagging"
"The BioMark™ HD System provides you with the ability to use a wide variety of sample types, multiple chemistry choices, and an industry leading degree of flexibility with no compromise in performance. The system offers unparalleled throughput for real-time PCR and digital PCR, integrating thermal cycling and fluorescence detection on integrated fluidic circuits (IFCs).
Integrated fluidic circuits significantly improve productivity in life science research through the automation of PCR reactions in nanoliter volumes. This is made possible by miniaturizing and integrating liquid handling components on a single microfluidic device. The BioMark HD System consists of a real-time PCR instrument, Dynamic Array™ IFCs for multiplexing samples and reagents, as well as an IFC loader to load the Dynamic Array IFCs."
Eight four-place fluidics stations
"Luminex xMAP technology is built on proven, existing technology-flow cytometry, microspheres, lasers, digital signal processing and traditional chemistry-that have been combined in a unique way. Featuring a flexible, open-architecture design, xMAP technology can be configured to perform a wide variety of bioassays quickly, cost-effectively and accurately.
Luminex color-codes tiny beads, called microspheres has 100 distinct sets. Each bead set can be coated with a reagent specific to a particular bioassay, allowing the capture and detection of specific analytes from a sample. Within the Luminex compact analyzer, lasers excite the internal dyes that identify each microsphere particle and any reporter dye captured during the assay. Many readings are made on each bead set, further validating the results."
"This is an advanced technology to process QIAGEN spin columns, enabling seamless integration of automated, low-throughput sample prep. All steps in the purification procedure are fully automated and up to 12 samples can be processed per run. It is designed around spin-column technology and includes an integrated self-positioning centrifuge, a heatable shaker and a pipette system with a robotic column gripper. The robot mimics exactly the same procedure from lysis, binding and washing through to the elution without a change in the chemistry or protocol."
Human Transcriptome Arrays
3' Expression Arrays
Gene Titan 96 samples
Gene Titan 24 samples
Gene Titan 16 samples
Affymetrix array hybridization and scan per GeneChip"
12.765 Digital Array
48.770 Digital Array
"Digital PCR works by partitioning a sample into many individual real-time PCR reactions; some portion of these reactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction of negative reactions is used to generate an absolute count of the number of target molecules in the sample, without reference to standards or endogenous controls."
"To meet the need for absolute quantitation and detection of target sequences Fluidigm qdPCR 37K™ IFC has the ability to perform four-plex detection. Since other platforms are limited to two-plex capabilities up to 80% savings per sample is expected when an experiment demands higher-plex capabilities, without sacrificing accuracy.
Key Features and Benefits of the Assay
• Rare Allele Detection
• Low-Fold Copy Number Discrimination
• Absolute Quantification of Viral Load
• Absolute Quantification of Nucleic Acid Standards
• Absolute Quantification of Next-Generation Sequencing Libraries"
"Automated Extraction from 0.2 ml BUFFY COAT
Automated Extraction from 0.3 ml BLOOD
Automated Extraction from 3.0 ml BLOOD
Automated Extraction from 10.0 ml BLOOD
Automated Extraction from 1.0 ml SERUM
Automated Extraction from 0.6 ml ORAGENE SALIVA
Automated Extraction from 4.0 ml ORAGENE SALIVA
Automated Extraction from 10 mg TISSUE
Manual DNA extraction per sample: other input options"
"Recent advances in nanofluidics technology have made possible the use of integrated fluidic circuits (IFCs) for high-throughput real-time PCR. Nanoliter-scale quantities of samples and reagents are channeled into thousands of nanoliter-scale chambers in which distinct real-time PCRs can be run. Fluidigm Dynamic Array integrated fluidic circuits (IFCs) deliver a new level of efficiency and throughput for real time PCR and genotyping that is compatible with existing TaqMan genotyping assays. Potentially, this technology allows up to 9216 individual TaqMan reactions to be run in a single experiment, with the promise of considerable reagent and time savings achievable from using nanofluidics arrays, compared with standard TaqMan genotyping on ABI PRISM 7900HT. Dynamic Array IFCs have an on-chip network of microfluidic channels, chambers, and valves that automatically assemble individual PCR reactions, decreasing the number of pipetting steps required by up to 100 fold. The available chip formats that can be used in our facility are:
• 48.48 - 48 samples x 48 assays (single use)
• 96.96 - 96 samples x 96 assays (single use)
Fluidigm SNPtype Assays can be used instead of Taqman assays that provide a custom, high- throughput, low-cost single nucleotide polymorphism (SNP) genotyping solution which enables rapid assay design and polymorphism screening. SNPtype Assays employ tagged, allele-specific PCR primers and a common reverse primer. A universal probe set is used in every reaction, producing uniform fluorescence while significantly reducing both startup and running costs. The assays provide excellent results on Dynamic Array IFCs and are compatible with existing workflows."
Human SNP Array 6.0 (Single Chip)
"The Array 6.0 features 1.8 million genetic markers, including more than 906,600 single nucleotide polymorphisms (SNPs) and more than 946,000 probes for the detection of copy number variation. The SNP Array 6.0 is the only platform with analysis tools to truly bridge copy number and association, including a new, high-resolution reference map and a copy number polymorphism (CNP) calling algorithm developed by the Broad Institute. The SNP Array 6.0 demonstrates industry-leading performance and represents more genetic variation on a single array than any other product, providing maximum panel power and the highest physical coverage of the genome.
The high price-performance value of the SNP Array 6.0 enables researchers to design association studies with larger sample sizes in the initial scan and replication phases, thereby significantly increasing the overall genetic power of their studies.
The SNP 6.0 Assay is based on a whole-genome sampling assay. In this assay, genomic DNA (500 ng) is digested with Nsp I and Sty I restriction enzymes and ligated to adaptors that recognize the cohesive 4 bp overhangs. All fragments resulting from restriction enzyme digestion, regardless of size, are substrates for adaptor ligation. A generic primer that recognizes the adaptor sequence is used to amplify adaptor-ligated DNA fragments. PCR conditions have been optimized to preferentially amplify fragments in the 200 to 1,100 bp size range. PCR amplification products for each restriction enzyme digest are combined and cleaned using a bead based or isopropanol precipitation method. Amplified DNA is then fragmented, labeled, and hybridized to the array."
Axiom on Gene Titan (16, 24 and 96 Format Array Plates)
"The Axiom Genotyping Solution is for genome-wide association studies (GWAS), replication studies, and candidate gene association studies. It includes predesigned and customized array plates with validated genomic content from the Axiom Genomic Database. This end-to-end solution also includes complete reagent kits, data analysis tools, and a fully automated workflow utilizing the Gene Titan Multi-Channel (MC) Instrument.
Key Features and Benefits of the Assay
• Flexible content - optimize your studies with a diverse set of relevant and novel variants
• Fast results
• More power - run more samples within your budget
• Greater confidence - generate reliable results with end-to-end automated sample processing and fully optimized reagent kits"
Contact facility for more information
Preparing STA Product for Sorted Cells (1-100 Cells per Well)
We can do Taqman assays and DELTAgene Assays. We do not do Primer Optimization.
"Recent advances in nanofluidics technology have made possible the use of integrated fluidic circuits (IFCs) for high-throughput real-time PCR. Nanoliter-scale quantities of samples and reagents are channeled into thousands of nanoliter-scale chambers in which distinct real-time PCRs can be run. Fluidigm DELTAgene Assays are high-quality, rapid turnaround, custom qPCR gene expression assays; also the platform is compatible with existing TaqMan Gene Expression Assays."
"The QuantiGene Plex (QGP) assay (Panomics/Affymetrix) is the most accurate and precise assay for multiplexed gene expression quantitation. Using the Luminex xMAP technology, QGP assays allow the simultaneous measurement of 3 to 80 mRNA species in every well of a 96-well plate. The QGP assay incorporates the exclusively-licensed and clinically proven branched DNA technology from Siemens that is used in the Versant® viral load assays for HIV, HBV and HCV. Branched DNA assays allow for the direct measurement of RNA transcripts by using signal amplification rather than template amplification. The assay is simple and easy to use; QGP does not require RNA extraction, cDNA synthesis or PCR amplification. The QuantiGene Plex assay is ideal to validate GeneChip or Next Generation Sequencing data and to validate biomarkers for translational research.
• True Multiplexing- Measure up to 80 genes of interest and housekeeping genes in the same well, with no cross-reactivity.
• Standardized Platform- 96-well plate format compatible with Luminex FlexMap3D systems.
• Simple Workflow- ELISA-like workflow for direct hybridization of transcripts to beads and transcript labeling.
• No PCR- No RNA purification, no reverse transcription, no PCR amplification and none of the associated artifacts.
• Works with Difficult Sample Types- Works with degraded and cross-linked RNA in FFPE tissues and directly with blood.
• Large inventory of validated genes- Over 15,000 genes can be mixed to create pathway- and disease-themed panels.
• Fast Customization- If Panomics don't have your gene(s), custom panels are available."
from PAXGENE BLOOD
We DO NOT offer RNA extraction service only; it is offered for projects with downstream applications done in our Facility."
Gene Expression Assay by Taqman
"Real-time detection of PCR products is made possible by including in the reaction a fluorescent molecule that reports an increase in the amount of DNA with a proportional increase in fluorescent signal. The fluorescent chemistries employed for this purpose include DNA-binding dyes and fluorescently labeled sequence specific primers or probes. Specialized thermal cyclers equipped with fluorescence detection modules are used to monitor the fluorescence as amplification occurs. The measured fluorescence reflects the amount of amplified product in each cycle.
Key Features and Benefits of the Assay
• One or Two step RT-PCR for RNA quantitation
• Plus/Minus assays using an internal positive control (IPC)"
"Taqman Genotyping Assay combines PCR amplification and detection in the same reaction. The method is based on the 5' nuclease activity of Taq DNA polymerase. A PCR is performed using primers that will amplify the DNA region containing the SNP of interest. Included in the reaction are two allele-specific fluorogenic probes, each consisting of a different fluorescent reporter dye and a fluorescent quencher. In the intact probe, the proximity of the quencher to the fluorphore causes fluorescence resonance energy transfer (FRET), reducing the fluorescence from the reporter dye. During PCR, the 5' nuclease activity of Taq digests the allele-specific probe bound to the region of the SNP, releasing the fluorescent dye from the quencher and allowing generation of a fluorescence signal. Depending on which dye signal is generated, the SNP alleles are determined. If only one dye signal is detected, the SNP is homozygous for the allele corresponding to the allele-specific probe, and if both dyes are detected, then the SNP is heterozygous."
"IPA is software that helps researchers model, analyze, and understand the complex biological and chemical systems at the core of life science research.
IPA helps you understand biology at multiple levels by integrating data from a variety of experimental platforms and providing insight into the molecular and chemical interactions, cellular phenotypes, and disease processes of your system. Even if you don’t have experimental data, you can use IPA to intelligently search the Ingenuity®Knowledge Base for information on genes, proteins, chemicals, drugs, and molecular relationships to build biological models or get up to speed in a relevant area of research. IPA provides the right biological context to facilitate informed decision-making, advance research project design, and generate new testable hypotheses."
Access is available to desktop sequence analysis software through the Penn Molecular Profiling Facility Bioinformatics Group.