Equipment, Services and Forms

We use real-time PCR to amplify and simultaneously quantify a targeted DNA/RNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific nucleotide sequences in a sample. The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in real-time.

Our CoRE facility is equipped with two ABI PRISM 7900HT sequence detection systems, configured to use 384-well or 96-well plates with SYBR green or TaqMan probes and capable of unattended 24-hour operation using a robotic arm to process plates in the queue. The facility has a QX100 Droplet Digital PCR System. It has many potential applications, including the detection and quantification of low levels of pathogens, rare genetic sequences, copy number variations, relative gene expression in single cells, and analysis of heterogeneous DNA methylation. The facility provides access to the Nanostring Technologies nCounter platform that allows for highly multiplexed (up to 800 targets per assay) digital quantification of nucleic acids. An eight channel 384/96-well format Beckman pipetting robot (Beckman Biomek NXp Laboratory Automation Workstation) is available for setting up PCR reactions in 384 and 96 well format. A Qiagen BioRobot Universal system is available for automated large-scale RNA and DNA isolation.

The following services are conducted using the ABI PRISM 7900HT.

  • Real-time PCR plate run (384- and 96-well plates)
  • Allelic discrimination run
  • qPCR SYBR-green and TaqMan assay design, synthesis, and validation
  • Allelic discrimination assay design, synthesis, and validation
  • TaqMan probe acquisition (synthesis only, 100 mmol conc.)
  • Reverse Transcription (RT)
  • SYBR-green and TaqMan real-time PCR assay setup (in triplicates)
  • Validated primer pairs for a large number of human, mouse, and rat genes
  • Genotyping

The ABI PRISM 7900HT sequence detection system (SDS) is designed for high throughput detection of fluorescent PCR-related chemistries. The ABI 7900HT is capable of real-time, end-point, and dissociation curve analyses. A computer coordinates the 7900HT instrument, bar-code reader, and automation module. The software saves the raw data from individual samples, whole plates, or batches of plates for analysis. More information on the instrument can be found here.

The instrument is currently configured to use 384-well or 96-well plates and is capable of unattended 24-hour operation using a robotic arm to process plates in the queue (384-well format is preferred because the cost of the actual plates is lower, as is the cost of reagents/reaction). Users drop off plates for assay after requesting services through iLab. Data will be available for download from the facility server. Login information will be provided to each customer upon registration with facility. Usual turnaround time is one working day. The SDS software from ABI is available from the core free of charge.

The Beckman Biomek NXp Laboratory Automation Workstation is an eight-channel 96/384-well format Beckman pipetting robot that utilized for setting up large-scale, real-time PCR reactions. Biomek NXp offers proven pipetting performance for low-volume (1µl – 20 µl ) PCR reaction setup with accurate and reproducible results.

The QX100 Droplet Digital PCR System from Bio-Rad is a third generation PCR system that can quantify RNA and DNA molecules using Droplet Digital PCR Technology. The equipment can be used for the detection and quantification of low-level pathogens, rare genetic sequences, copy number variations, relative gene expression in single cells, and analysis of DNA methylation.

The QX100 system consists of two components: the QX100 droplet generator and the QX100 droplet reader. The QX100 droplet generator partitions samples containing cDNA or RNA template into 20,000 nanoliter-sized droplets. After PCR on a standard thermal cycler, droplets from each sample are streamed in single file through the QX100 droplet reader. The PCR-positive and PCR-negative droplets are counted to provide absolute quantification of target DNA in digital form.

In addition to standard Nanostring assays we provide mRNA, lncRNA, elements, ChIP-string, microRNA, and copy number variation (CNV), as well as Nanostring cartridge scanning usage using a Nanostring nCounter Gene Expression (GEN2) Analysis System.

The nCounter system from Nanostring Technologies is a complete, fully automated system for the next generation of digital gene expression analysis. The nCounter is designed to provide an ultrasensitive, reproducible and highly multiplexed (up to 800 genes) method for detecting gene expression across all levels of biological expression. This assay provides a method for direct measurement of mRNAs and miRNAs with molecular barcodes called nCounter Reporter Probes without the use of reverse transcription or amplification. Since the system literally counts the individual nucleic acid molecules, it is also used for CNV analysis, ChIP analysis (ChIP-string), and single cell gene expression analysis. The assay can be run on total RNA isolated from any source, including formalin-fixed paraffin embedded (FFPE) samples.

The complete nCounter system includes two instruments. The nCounter PrepStation (liquid handling robot) is used to purify ternary complexes obtained during hybridization, to remove unhybridized probes and to immobilize the complexes into the cartridge for subsequent image acquisition. The nCounter Digital Analyzer (image acquisition instrument) is used to scan the cartridge and generate raw data (counts). For further information on the capabilities of the instrument and technology, please visit here.

We provide both  DNA/RNA extraction from whole blood, tissues and FFPE samples and

DNA/RNA extraction using a Qiagen Universal Biorobot (cells only).

The equipment we use for these processes is a Qiagen BioRobot Universal system. This integrates all the instrumentation, software, purification technologies, and enzyme technologies that are required for high-throughput purification of highly pure RNA and genomic DNA from cells and plasmid DNA from bacteria. RNA can be purified from cells using the RNeasy 96 BioRobot 8000 kit in multiples of 96 up to 192 samples per run.

We offer an introduction to Real Time PCR and Nanostring resources.

Real Time PCR

You may explore Real Time PCR through two common methods, the first of which is fluorescent dyes (e.g. SYBR green) that intercalate with any double-stranded DNA. The second; uses- sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target (e.g. TaqMan assays).

We recommend the use of SYBR green for detection. There are several advantages of using SYBR green. Multiple reactions can be set up rapidly and inexpensively with standard oligonucleotides. SYBR green will detect any PCR product, including nonspecific products and primer-dimers. Therefore, care must be taken during oligonucleotide design. The qPCR core has a collection of large number of primer pairs for human, mouse and rat genes. Our primers are 20-mers with 55% G-C content and a single 3' -G or -C. We tested the majority of our primer pairs for specificity and absence of primer-dimer formation by PCR followed by gel electrophoresis.

We also routinely use other detection systems such as molecular beacons and Invader and TaqMan assays. Although commercial kits are available, we use our own design, assembling our own reaction components. For details on real-time PCR protocols, visit PubMed to review these studies:

  1. Monitoring G-protein-coupled receptor signaling with DNA microarrays and real-time polymerase chain reaction. Methods Enzymol. 2002. Vol.345: 556-69. PMID: 11665639
  2. Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays. Nucleic Acids Res. 2002. Vol.30: e48. PMID: 12000853

Nanostring

NanoString nCounter technology is a direct digital detection system, which enables highly sensitive and reproducible multiplexed gene quantification without amplification. It can simultaneously analyze up to 800 genes in a single tube reaction. The technology is used for the detection of gene expression signatures, DNA copy number assessment, miRNA and lncRNA profiling, gene fusion analysis, DNA looping, nCounter vantage assays, and chromatin immunoprecipitation analysis (ChIP-String). The technique can directly assay tissue and blood lysates as well as FFPE extracts in addition to purified RNA and DNA samples. Learn more about the potential applications of this technology.