Gene Expression Analysis

The ability to monitor changes in gene expression has been critical in identifying the causes or development of many diseases, especially cancer. Gene expression profiling or microarray analysis has enabled the measurement of thousands of genes in a single RNA sample. There are a variety of microarray platforms that have been developed to accomplish this and the basic idea for each is the same: a glass slide or membrane is spotted or "arrayed" with DNA fragments or oligonucleotides that represent specific gene coding regions. Purified RNA is then fluorescently labeled and hybridized to the slide/membrane. In some cases, hybridization is done simultaneously with reference RNA to facilitate comparison of data across multiple experiments. After thorough washing, the raw data is obtained by laser scanning. The data may then be entered into a database and analyzed by a number of statistical methods. Typically, researchers utilize expression arrays to investigate global gene expression patterns and use quantitative PCR to confirm and individually monitor the gene or genes associated with the particular condition or disease.

Empire Genomics can assist in determining global patterns of gene expression alterations through expression arrays from several instrument platforms, or precisely monitor expression of several genes known to be involved in a disease via quantitative PCR. Illumina and Agilent expression arrays are available. Agilent microarrays are manufactured using a proprietary non-contact industrial inkjet printing process, in which oligo monomers are deposited uniformly onto specially-prepared glass slides. This in situ synthesis process prints 60-mer length oligonucleotide probes, base-by-base, from digital sequence files. The Illumina Expression BeadChip provides genome-wide transcriptional coverage of well-characterized genes, gene candidates, and splice variants, delivering high-throughput processing of 12 samples per BeadChip without the need for expensive, specialized automation. Illumina has whole-genome expression arrays for intact RNA samples and DASL arrays for partially degraded or difficult to obtain samples.

Real time PCR analysis is available to quantitatively analyze gene expression of one to five genes of interest. Platforms available include ABI Taqman and BioRad MyIQ. Individual samples or 96 or 384 well plates can be analyzed. Predesigned or custom designed probes can be utilized. Differences among Real-Time PCR kits are largely restricted to the DNA polymerases and the detection methods used. Linear sequence-specific oligo probes, commonly known as TaqMan probes, bear a fluorescent reporter dye at the 5’-end and a quencher molecule at the 3’-end. Usually, they are designed to anneal at a sequence slightly downstream of one of the PCR primers. Since fluorescence dye and quencher are in close proximity, the fluorescence is quenched and not detectable. As soon as the Taq polymerase reaches the 5’-end of the probe it cuts-off the fluorescence dye utilising it’s 5’-3’ nuclease activity, thereby releasing the fluorescent reporter into solution. The resulting increase in fluorescence can be measured to monitor the amplification of the target DNA. These probes are very specific, however, they are rather expensive and a new probe has to be designed for every new target DNA. If extra specificity is not necessary, one may circumvent these drawbacks by switching to Real-Time PCR assays based on fluorescent DNA-binding dyes such as SYBR Green I (BioRad). The latter intercalates non-specifically into double-stranded DNA which leads to a thousand-fold increase of the fluorescence signal. Since the amount of DNA is boosted with every new cycle, the fluorescence increases proportionally and can be monitored to measure the amplification of the target DNA. Empire can assist in selection of the array and expression analysis platform that is best for your needs as well as sample quality and quantity analysis and labeling.