Overview of Analytical Services at UC Riverside CIF
1. Genomics Suite
DNA Sequencing
Sequencing of cloned inserts or PCR products is done by combined DNA polymerase-mediated linear amplification of templates in the presence of mixtures of dNTPs and fluorophore-labeled dideoxynucleotide triphosphates (otherwise known as Sanger sequencing or dideoxynucleotide chain termination sequencing), followed by fractionation of the extension products by capillary electrophoresis. We use a 96-capillary instrument, an Applied Biosystems 3730xl. The 3730xl typically yields read lengths of 800 bases; 900 bases or more are attained with the best quality templates. Please see Guidelines for Template Preparation for information on purifying template DNA, submitting your templates, retrieving sequences and viewing text and electropherogram files.
Turnaround time. We do sequencing reactions 5 times a week. In most cases, data will be posted to our server on the second working day after templates are received; for instance, if you bring templates in on Monday by 3:45 p.m., data will be posted by Wednesday noon. Emails are sent out upon posting to inform clients that data is ready for download. We provide password-protected login to our server.
Handling libraries preparatory to sequencing or PCR reactions for microarray making. We provide services for clients wishing to process genomic or cDNA libraries for sequencing. We automate colony picking from conventional size petri dishes or large format culture plates (22 cm square, holding up to 2000 colonies comfortably) with our Genetix QPIX. Bacterial cell cultures in 96-well format are incubated at 37 deg overnight, with aeration and shaking at 500 rpm, in our GeneMachines Hi-Gro tower incubators. Plasmids are extracted with a conventional alkaline lysis procedure and isolated in 96-well format from lysate by vacuum filtration and adsorption/desorption on fibrous silica, making use of our 96-channel Beckman Multimek and our 8-probe MWG Roboprep 2500. For optimizing conventional or DNA sequencing PCR reactions, DNA (plasmid) yields can be quantified with PicoGreen and our plate-reading fluorometer, the Packard Fusion, which is integrated with a second liquid-handling robot a Packard Multiprobe II. A second option available is use of our Nanodrop spectrophotometer, a cuvetteless instrument requiring only 1.5 microliters sample. We provide training in use of the QPIX (for those needing colony picking or macroarray making capability), Nanodrop and Fusion, and clients pay a fee for their use, while the Roboprep 2500 and Multiprobe II are operated by CIF personnel only.
Sequencing by Primer Walking. We can sequence large insert plasmids or cosmids by primer walking. We need a few micrograms of template (please call Yoon Gi Choi at 23984; ychoi@ucr.edu) and information on the identity of the vector. We will design primers, do the sequencing reactions and fractionate the extension products. Additional rounds of primer design and sequencing takes us through your template. We post sequencing results on our server for download, give you the primers, assemble the final sequences and provide a restriction map. There is an initial $10 charge for setup but after that the investigator pays only the cost of the primers and the sequencing reactions at our normal rate ($6.30 per reaction).
2. Gene Expression Suite
Expression studies with commercial arrays.
Equipment. CIF has equipment from Affymetrix for hybridization (Hybridization Oven 640), staining and washing (Fluidics Station 450) and scanning (Scanner 3000) of stained arrays. Our acquisitions, in Nov. 2003, were after Affymetrix redesigned their GeneChips®, reducing feature size from 18 to 11 microns to increase the number of features per array. Our new scanner is capable of scanning at the increased resolution necessary for the smaller features. The scanner was upgraded in June 2005 to scan at sub-micron resolution, giving it the capability to scan the newer 5 micron tiling arrays.
Workflow. Minimal sample requirement for GeneChip® analysis is 16 micrograms total RNA at a concentration of at least 1 microgram/microliter. Please inquire about options for amplifying much smaller amounts of starting material. Our full service rate includes, preparatory to hybridization, RNA quantitation with a Nanodrop cuvetteless spectrophotometer, quality check with an Agilent Bioanalyzer 2100, a chip-based, rapid analysis by capillary electrophoresis, 1st and 2nd strand cDNA synthesis and in vitro transcription/amplification with biotin labeling. Hybridization to GeneChips® is followed by staining and amplification of the signal are done according to well-established protocols worked out by Affymetrix.
Scanning and data analysis. Arrays are scanned in the Affymetrix environment GCOS (GeneChip Operting System) to generate the pixelated ".dat" files, which are then processed to create the single intensity ".cel" files. Probe sets representing expressed genes are then statistically analyzed to generate numerical indicators of expression levels for each gene, that can be compared between experimental groups, and that are annotated with information contained in Affymetrix "library" files in creating a ".chp" file. These files represent data processed with the Affymetrix algorithm, MAS5, and they can be exported from GCOS to a ".txt" file which can be opened in GCOS or Excel. Data analysis with GCOS stops with the .chp file. For investigators wanting to do their own first level analysis, .dat, .cel and .chp files are assembled into a single .cab or .dtt file and delivered on a CD or transferred by ftp. GCOS v. 1.3 is available at no charge to GeneChip® users, and CIF recommends downloading this software from the Affymetrix web site. Manuals and tutorials are also available on the Affymetrix web site. Investigators with .dat files produced with Microarray Suite can use GCOS for first level analysis (files are imported, but not directly opened with GCOS). Worthy options for higher level data analysis include GeneSpring from Silicon Genetics (now a part of Agilent) and Bioconductor project, an open source package of analysis tools in R that is maintained by Thomas Girke of UCR's Bioinformatics core. In CIF, we maintain and provide training in use of freeware data analysis tools from The Institute for Genome Research (TIGR; MIDAS, MEV) that are especially suited for use with data from two-color arrays. In addition we maintain a single computer license for ArrayAssist 4.1.1 from Stratagene, and we provide training in its use; this program contains a complete set of preprocessing algorithms suited for use with one-color Affymetrix arrays, as well as filtering, clustering, data mining and discovery tools.
Preparation of spotted microarrays and scanning after hybridization. The instrument for making arrays in CIF is the VersArray ChipWriter Pro from Bio-Rad (originally engineered for Virtek). This precision instrument is capable of producing arrays with up to 50,000 spots per slide. Image files are acquired with a Perkin Elmer ScanArray Express with three lasers. We provide a workstation for spotfinding and first level analysis with Imagene 5.5 (Biodiscovery).
Quantitative PCR Many investigators will want to quantify specific transcripts, sometimes as follow-up or validation of findings obtained with expression arrays. For this purpose CIF has an Applied Biosystems 7700 Sequence Detection System and a newer Bio-Rad iCycler/iQ5 for quantitative PCR. These instruments allow determination of starting copy number of individual transcripts or cDNAs, in absolute quantitation or fold-change in gene of interest when normalized to reference genes in relative quantitation. PCR reactions are done in 96-well PCR plates or strips of tubes and amplicon is quantified cycle by cycle with the use of fluorophore-labeled oligonucleotide probes (containing reporter and quenching fluorophores) or with the use of an intercalating fluorescent dye, SYBR Green I. See Yoon Gi Choi (ychoi@ucr.edu; ext. 23984) in CIF for consultation on sample preparation, experimental design, choice of reagents and training in use of our ABI 7700 and/or Bio-Rad iQ5. Workshops and instrument demonstrations are done on a regular basis. Additional information can be found at:
UCR CIF Home Page Please contact Yoon Gi Choi (ychoi@ucr.edu) if you have enquiries.