SCGC Gene Expression
The SCGC operates an Illumina BeadLab 1000, a LIMS supported laboratory system capable of processing custom snp, whole genome snp genotyping, methylation profiling as well as custom and whole genome gene expression profiling.
In support of our gene expression services the SCGC has recently invested in an iScan scanner, additional Tecan robotics and a SciGene little dipper array processing robotic workstation. These capital improvements have resulted in a greatly increased expression chip processing capacity as well as increased reproducibility and consistency. The facility is capable of running all current Illumina products designed for the BeadArray or iScan readers and can process up to 288 expression samples per day.
Illumina whole genome gene expression profiling protocols begin with 100 ng of total RNA isolated from whole blood or tissue. All samples are quanted using a Ribogreen fluorescent assay and normalized to 10 ng/ul prior to amplification. Amplified and labeled cRNA is produced using the Illumina specific Ambion TotalPrep kit. 1st and 2nd strand cDNA is produced using the Ambion kit and purified using a robotic assisted magnetic capture step. Biotinylated cRNA is produced from the cDNA template in a reverse transcription reaction. Typical yields are in excess of 1.5 ug. After a second Ribogreen quant and normalization step, amplified and labeled cRNA is hybridized overnight at 58C to the expression arrays. Hybridization is followed by washing, blocking, staining and drying on the Little Dipper processor. Array chips are scanned on either the Beadarray reader or the iScan reader. Expression data is extracted and compiled using BeadStudio software ( Illumina) and extracted( un-normalized) data as well as raw scan data is delivered to end users on DVD
The SCGC currently supports all Illumina gene expression whole genome products including Human and Mouse Ref6 and Ref8 chips, RatRef12 and Human HT-12 chips. In addition we support DASL and GoldenGate custom expression profiling.
