Path BioResource welcomes back Penn researchers!

100% operations restored for FY22, effective 7/6/2021.

Please contact PBRHELPDESK@pennmedicine.upenn.edu re: questions or concerns.

AUTOMATED NUCLEIC ACID EXTRACTIONS

DNA: MSM I (CHEMAGEN)

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.


Chemagen MSM I

RNA: QIACUBE (QIAGEN)

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.


QIAcube

SNP GENOTYPING

Genotyping provides a measurement of the genetic variation between members of a species of which Single Nucleotide Polymorphisms (SNP) are the most common type of genetic variation. A SNP is a single base pair variation at a specific locus, usually consisting of two alleles (where the rare allele frequency ≥ 1%). SNPs are often found to be the etiology of many human diseases and are becoming of particular interest in pharmacogenomics. Because SNPs are evolutionarily conserved, they have been proposed as markers for use in association studies in place of microsatellites. The use of SNPs is extended in the HapMap project to provide the minimal set of SNPs needed to genotype the human genome. SNPs can also provide a genetic fingerprint for use in identity testing.



SINGLE ASSAY: Quantstudio 12K Flex (LIFE TECHNOLOGIES)

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.

The ABI reactions contain 5 ng of genomic DNA, specific PCR primers, two allele-specific probes, dNTPs, Taq DNA polymerase and buffer according to the manufacturer's specifications. Reactions are set up in 384-well plates using a Biomek FX (Beckman Coulter), and cycled in ABI 7900 HT. SNP alleles are called using ABI software that provides scatter plots of allelic calls.


Quantstudio 12K Flex

MULTIPLE ASSAYS: BIOMARK HD (FLUIDIGM)

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 96.96 Array

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.

GENOME WIDE ASSAY

Affymetrix is a leading provider of microarray solutions for targeted and genome-wide applications. Their powerful portfolio includes arrays, reagents, instruments, and informatics tools that enable you to detect common and rare single nucleotide polymorphisms (SNPs), copy number variants, and other genetic variations that can contribute to complex diseases.

MICROARRAY ASSAY: GENE TITAN (AFFYMETRIX)

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

Gene Titan Multi Channel Instrument

MICROARRAY ASSAY: GENECHIP INSTRUMENT SYSTEM (AFFYMETRIX)

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.


Genome-Wide Human SNP 6.0 Array

AGILENT aCGH ASSAY: CGH & CGH+SNP Microarrays

Agilent CGH microarrays are commonly used for genome-wide chromosomal analysis. Agilent offers catalog products for a variety of CGH applications as well as most affordable and flexible custom content options.

Comparative Genomic Hybridization (CGH) is a technique used for measuring copy number changes over an entire genome between two DNA samples—a sample and a control. With the copy number information one can visualize large or small chromosomal aberrations, including very small DNA segments such as microdeletions and microduplications. Using Agilent microarray technology based on 60-mer oligo array probes the genome can be scanned for imbalances on a massively parallel scale, thus increasing the coverage and resolution compared to other microarray technologies.

Agilent's 2-color CGH array provides superior copy number data as opposed to 1-color because sample and reference DNA are directly compared on the same array. Agilent's long 60-mer probes are more specific than probes that are shorter in length, allowing one to use fewer probes to make accurate calls.

Agilent offers three types of cytogenetic microarrays: CGH, CGH+SNP, and CNV, which are optimized for the most challenging sample types including FFPE and single cells. The microarrays are available for human, mouse, rat and other model organisms. The targeted microarrays include those designed for exon level coverage of various disease associated and cancer associated regions of the genome.


Agilent Scanner

GENE EXPRESSION

Gene expression is the most fundamental level at which the genotype gives rise to the phenotype. The genetic code stored in DNA is "interpreted" by gene expression, and the properties of the expression give rise to the organism's phenotype. Such phenotypes are often expressed by the synthesis of proteins that control the organism's shape, or that act as enzymes catalyzing specific metabolic pathways characterizing the organism. In general gene expression is regulated through changes in the number and type of interactions between molecules that collectively influence transcription of DNA and translation of RNA.




REAL TIME PCR

In conventional PCR, the amplified product, or amplicon, is detected by an end-point analysis, by running DNA on an agarose gel after the reaction has finished. In contrast, real-time PCR allows the accumulation of amplified product to be detected and measured as the reaction progresses, that is, in "real time".

SINGLE ASSAY: Quantstudio 12K Flex (LIFE TECHNOLOGIES)

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)

Quantstudio 12K Flex

MULTILPLE ASSAYS: BIOMARK HD (FLUIDIGM)

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.

MULTIPLEXED ASSAY: FLEXMAP 3D (LUMINEX)

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.

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.

Luminex xMAP 3D

MICROARRAY ASSAY: GENE TITAN (AFFYMETRIX)

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

GeneChip Array Plate

MICROARRAY ASSAY: GENECHIP INSTRUMENT SYSTEM (AFFYMETRIX)

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.


Affymetrix GeneChip System

DIGITAL PCR

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.

MULTIPLE ASSAY: BIOMARK HD (FLUIDIGM)

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

Fluidigm System

MULTIPLEXED AMPLICON LIBRARY PREPARATION FOR NEXT GENERATION SEQUENCING

ACCESS ARRAY SYSTEM (FLUIDIGM)

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