Equipment

Ion Torrent Personal Genome MachineIon Torrent Personal Genome Machine

The Genomics Facility Core (GFC) has obtained an Ion Torrent Personal Genome Machine (PGM; pictured) to implement next-generation sequencing capability within the Southwest Environmental Health Science Center. In the GFC's first project with the PGM, tumor samples from melanoma patients were sequenced for mutations in five hotspots in their genome (B-Raf codon 600; N-ras codons 12 and 13, and codon 61; and c-Kit codons 576 and 642). The sequence data shows two different mutations in codon 61 (shown by blue highlighted nucleotides and resulting in Q to R and Q to K amino acid mutations shown above the nucleic acid sequence) of the N-ras gene in the first two patients sequenced by the GFC using the PGM. Knowledge of which mutations are present in a patient's tumor will alter treatment decisions in the future as the technological capability of the GFC is translated into the clinic.

 

Sequenced Data

 

cDNA and oligonucleotide microarrays

The GFC performs the RNA quality control, fluorescent labeling, hybridization, scanning, quantitation, and analysis steps of the two-color microarray process utilizing spotted microarrays manufactured on-site. The GFC offers human and mouse cDNA microarrays consisting of 5,760 elements with ~3,000 of the elements representing known genes. The sequences printed on the cDNA microarrays are derived from sequence validated bacterial clones. The cDNAs are amplified, purified, quantitated, and checked for correct size by agarose gel electrophoresis before being robotically spotted onto Corning’s UltraGAPS II slides using a GeneMachines OmniGrid printer and Stealth printing tips from Telechem International. The human 20,600 oligonucleotide array is also manufactured using the OmniGrid and TeleChem printing tips from a library synthesized by Sigma and designed by Compuge

RNA sample quality control

The GFC analyzes and quantitates all RNA samples before use in microarray hybridization and real time PCR using the Agilent Bioanalyzer. The samples are loaded into the microwells of a Bioanalyzer chip, the sample is separated by size in the chip and analyzed using a fluorescent dye as the RNA passes a detector. The results can be used to detect and quantitate RNA degradation in the sample. A major advantage of the Agilent Bioanalyzer is the small sample requirement –typically 100-200 nanograms of RNA are consumed, which is a benefit to users with limited RNA quantities. In addition, the GFC can use the results to calculate a RNA Integrity Number (RIN) that allows quantitative comparison of RNA sample quality and provides a threshold value for usable samples. This can prevent expensive analyses being performed on samples of poor quality that could lead to unreliable results. 

 

Affymetrix GeneChip System

Affymetrix GeneChips are commercially manufactured oligonucletide arrays made by directly synthesizing oligonucleotides on a silicon chip using photolithography. Users supply sample RNA and the GeneChip for analysis. The Genomics Technology (GFC) performs all subsequent steps of the microarray process including data analysis and data archiving. Results are uploaded to the GFCs password-protected web site. The Affymetrix platform offers whole genome coverage on a single array of the three primary model systems of SWEHSC investigators: human, mouse, and rat. The Affymetrix GeneChip platform does not use a two-channel hybridization scheme common to the microarrays manufactured on-site by the GFC. Therefore, in order to acquire differential expression results, it is necessary to hybridize at least two GeneChips one for the test and one for the control. An advantage of the single color system over the two-color system is realized, however, when multiple test samples are run. Since the test hybridizations are independent from the control hybridization, only one control sample has to be prepared regardless of the number of test hybridizations. The Affymetrix GeneChip system has also been upgraded in the last two years to allow researchers to utilized the new generation of high density Affymetrix GeneChips: 500k Single Nucleotide Polymorphism Chips, Promoter tiling Chips, and Genome tiling Chips.

 

Real-Time PCR

After performing microarray analysis, many GFC users wish to confirm the results. To meet this need, the GFC offers gene expression analysis using the Applied Biosystems Prism 7000 and 7300 sequence detection systems in conjunction with TaqMan Real-Time PCR Assays-on-Demand or Assays-by-Design from Applied Biosystems. The user provides RNA (often this can be the same RNA used for the microarray analysis) along with the relevant primers purchased through Applied Biosystems Assays-on-Demand or Assays-by-Design programs. The GFC will reverse transcribe and amplify the sample RNA and analyze the results. The GFC provides consultation on experiment design and parameters, as well as help in ordering primers. The GFC also offers use of the real time PCR machines to users that want to set up and analyze their own real time PCR reactions. As an alternative to the high cost of TaqMan primer/probe sets, the GFC also offers a low-cost alternative using a Universal Probe Library of locked nucleic acids from Roche Applied Science.

Because the real-time PCR reaction run by the GFC is comparative, a reference gene is used to correct for technical error such as variance in the amount of RNA sample used. A common reference gene used to measure baseline expression is the GAPDH, however, some users may want to use another gene (e.g. Histone 3.3, beta-actin, or beta-glucuronidase) that better suits their experimental model. Since the ABI 7000 and 7300 are able to accommodate up to 96 reactions at once, the reference reactions are run in parallel with the test gene reactions.

GE HealthCare Codelink Bioarrays

Codelink Bioarrays are manufactured by spotting 15-mer oligonucleotide probes onto a glass slide coated with a 3-dimensional polyacrylamide gel matrix. Each gene on the array is represented by oligomer pre-tested for sensitivity and specificity. As with the Affymetrix GeneChip system, Codelink sample preparation is different from cDNA array sample preparation. RNA is first reverse transcribed with a T7-oligo(dT) primer followed by double strand synthesis. The double stranded cDNA is purified and used in an in-vitro transcription (IVT) using T7 RNA polymerase and biotinylated ribonucleotides, resulting in a biotinylated cRNA product. Following hybridization, the Bioarray is washed, stained with streptavidin labeled Cy3, and scanned to obtain data. Like the Affymetrix platform, the Codlink system uses single color hybridization. The Codelink Bioarrays provide single array, whole genome coverage of human, mouse, and rat model systems with greater sensitivity than the Affymetrix GeneChip system at a comparable price.

CpG island microarrays

The GFC has developed a new type of microarray to query genomic elements for CpG methylation levels. The GFC has printed arrays of genomic DNA containing CpG islands. Abnormal methylation of these islands represents one of the early events in tumorigenesis and is associated with silencing of a number of tumor suppressor genes including MASPIN, MGMT, and p16. Using the new CpG Island microarrays, researchers are able to scan samples for genes silenced by abnormal methylation in human cells.

 

Affymetrix Fluidics station

Axon 4000b dual color laser scanner

ArrayBooster automated hybridization station

Affymetrix automated hybridization oven