Functional Genomics - Services
Next Generation Sequencing: In collaboration with the Rutgers University Cell and DNA Repository (RUCDR), our shared resource has access to multiple sequencing platforms to meet the needs of CINJ researchers. These include: Illumina HiSeq2500, Illumina MiSeq, Ion Torrent PGM and Ion Torrent Proton. The services provided will vary depending on which instrument is used for the particular analysis performed. The appropriate choice of instrument and experimental approach will be determined by the investigators in consultation with staff from the Functional Genomics, Biometrics, and Bioinformatics shared resources, who will be engaged in all stages of experimental design, data acquisition, processing and analysis.
DNA and/or RNA extraction from tissues, cell lines, or chromosome immunopreciptations for downstream sequencing applications can be performed manually by the investigators themselves for small sample sets or by using automation (QiaSymphony) for larger sample sets and higher throughput.
Following submission, the shared resource will perform quality analyses on each sample. Sample preparation for the sequencing platforms, including library preparation, emulsion PCR, etc., are highly technical processes which must be performed correctly to ensure adequate sequence reads. These services will be included in the cost of sequencing and will be performed by shared resource staff. DNA/RNA library preparation is carried out via automation using the Caliper SciClone system to ensure quality and consistency across any sample set.
Raw data from the sequence analysis will be pre-processed to ensure quality control and transferred to the Bioinformatics shared resource for detailed analysis (including functional analysis) to elucidate the underlying biological processes giving rise to the observed data. Processed data will also be stored on one terabyte external hard drives provided to the investigators.
Analysis data can be validated through follow up with QPCR. We have multiple systems available to CINJ researchers for this purpose, including: Stratagene Mx3005P, Life Tech ViiA™ 7 Real Time PCR Instrument and Fluidigm (BioMark EP1 and C1).
DNA Microarray: The Functional Genomics Shared Resource supports Affymetrix GeneChip technology for monitoring global gene expression using commercially available arrays capable of analyzing expression changes across entire genomes. Affymetrix expression arrays are available for gene sets from human, rat, mouse, Arabidopsis, Drosophila, yeast, E. coli and other organisms. In addition, RNA extraction and quality analysis services are available and use a 2100 Bioanalyzer from Agilent Technologies, as well as NanoDrop ND-1000, to assure the quality and quantity of RNA before labeling. The shared resource offers comprehensive microarray analysis including RNA extraction and quality testing, sample clean-up, linear amplification (where applicable), labeling, hybridization, scanning, preliminary image analysis, quality analysis, data management, data storage and conservation and experiment analysis.
In addition, as a way to validate microarray expression results, we now offer real-time PCR services using the Agilent Technologies Stratagene Mx3005P. To access these services, the requester submits purified total RNA samples. Protocols for preparing RNA can be accessed via the following links: http://tools.invitrogen.com/content/sfs/manuals/trizol_reagent.pdf
An RNA quality check using the Agilent 2100 Bioanalyzer is performed and RNA concentrations determined by the NanoDrop ND-1000 spectrophotometer. Experimental samples are submitted with a control sample for comparison.
A comprehensive quality control program is used by the facility. Every GeneChip or spotted array contains performance features, which are examined, compared with industry and baseline criteria and recorded. Data from arrays or GeneChips that do not meet rigorous pre-determined performance standards are repeated.