Gene Expression Assays: RNA-Seq, QPCR and Microarrays

Gene expression assays measure which genes are expressed in different samples, or how the expression of genes change over time or in response to specific treatments. The ATG Shared Resource has three types of assays for measuring gene expression: RNA-Seq, Real-Time PCR and Affymetrix GeneChips.

Next-Gen Sequencing and RNA-Seq

RNA-sequencing (RNA-seq) is a next-generation sequencing approach to measuring gene expression across all the genes at once. Briefly, RNA is isolated, fragmented, converted to cDNA then ligated to adaptors and sequenced. The resulting sequencing "reads" are aligned to the genome. The number of reads aligned to each gene is proportional to the gene expression level. RNA-seq may also provide information about mutations (e.g. SNPs) and alternative RNA splicing, and chromosomal translocations can be detected by analyzing the output for the presence of fusion transcripts that cross the translocation breakpoint. RNA-seq can be successfully performed with high-quality RNA from fresh or frozen tissues or cell lines, and with RNA isolated from pathology samples, such as Formaldehyde-Fixed, Paraffin-Embedded (FFPE) material.

TaqMan Real-Time PCR Assays

Quantitative Real-Time PCR, or QPCR, measures increases in the production of PCR products during the PCR reaction, using a fluorescent assay. QPCR is the standard for highly accurate measures of RNA or DNA levels in a sample. The ATG Shared Resource specializes in the use of Applied Biosystems TaqMan QPCR assays, which use three probes per PCR product for enhanced specificity.

QPCR assays are most appropriate for performing highly accurate measurements of fewer than 100 genes at a time. The ATG Shared Resource performs QPCR assays in 384-well format to save reagents and keep costs lower.

Affymetrix Gene Expression Assays

Affymetrix GeneChips are the gold standard for measure gene expression across all genes at the same time. There are several different types of Affymetrix assays available, including 3'-end arrays, microRNA arrays and all exon arrays.

Affymetrix GeneChips have multiple probes for each gene and include many built-in controls, so the data is very high quality. The ATG Shared Resource provides expert data analysis of Affymetrix gene expression experiments performed in the facility, using a variety of sophisticated software tools and approaches.

Please contact the ATG Shared Resource staff for more information.

Technical Director:
Gavin Pickett, Ph.D.
CRF G-21
Tel: (505) 272-0680

Jason Byars
CRF G-21
Tel: (505) 272-0675

Senior Technical Staff:
Jamie Padilla, M.S.
CRF 118
Tel: (505) 272-5564

Jennifer Woods
CRF 121
Tel: (505) 272-3464

Faculty Director:
Scott A. Ness, Ph.D.
Professor, Internal Medicine
Office: CRF 121; Tel: (505) 272-9883