Protocols
The Genomics Core Facility supplies custom arrays to individual laboratories. The sections below explain the protocols followed throughout this process in great detail including techniques for preparing samples, printing clones, hybridization and quantification of results.
Preparation of cDNA Clones
The cDNA clones are selected from the I.M.A.G.E. consortium of cDNA clones available at the NCBI website. These clones can then be purchased from Research Genetics Inc and other companies. The clones are received as bacterial cultures. The DNA microarray core processes these clones to produce PCR products using generic primers supplied by Research Genetics. If required the PCR reactions are optimized to produce single bands. To prepare cDNA clones for printing/arraying, large-scale PCRs are performed with the help of Biorobot 3000, a liquid handling robot with an integrated PCR machine and spectrophotometer set-up. This enables not only the automation of the PCR procedure, post-PCR purification of samples (to eliminate primers and primer-dimers) and quantitation of the products but also eliminates human error during these steps. For quality control purposes, aliquots of the PCR products are analyzed at different steps during this procedure. The cDNA are quantitated and normalized to a desired concentration and resuspended in a suitable buffer for printing/arraying.
See a PowerPoint schematic of the methodology for preparation of clones for arraying. [link ppt file]
Printing/Arraying of cDNA Clones
The DNA microarray core uses the Flexys microgridding robot. The DNA microarray core is currently using glass slides coated with super-amine substrates for printing. The super-amine substrate (Telechem, Inc.) provides a highly favorable surface for binding DNA uniformly. The cDNA clones ready for printing are transferred to 384-well plates in a specific fashion resulting in a desired pattern. All clones are printed in duplicate. All arrays have the ability to detect certain spiked-in controls that serve as positive controls. The cDNA arrays are UV-crosslinked to the substrate, denatured and stored for future use.
Sample Preparation
For microarray experiments total RNA is prepared using the RNAeasy kit (Qiagen Inc.) following the manufacturer’s protocol. If a trizol-based protocol is used for preparing total RNA, RNAeasy kit is used to purify the sample further using the RNA clean-up protocol. The quality of RNA is checked on a gel before using it for microarray experiments.
Sample Preparation for miRNA Microarrays
For microarray experiments, total RNA that includes miRNA may be prepared by using the miRNeasy Mini Kit (Qiagen, Inc.) following the manufacturer’s protocol. The quality of RNA is checked using the Agilent Bioanalyzer.
Sample Preparation for NimbleGen CGH Arrays
Genomic DNA may be isolated using customer preferred methods. The DNA will need to be sonicated prior to submission to the Genomics Core Facility. The fragment sizes should range from ~500 bp to 2000 bp, with the majority of fragments in the 500 bp to 1000 bp region.
Sample Preparation for NimbleGen ChIP-on-Chip Arrays
Sample preparation may be accomplished by following NimbleGen’s ChIP-on-chip protocol (pdf). [link pdf file]
Sample Preparation for NimbleGen DNA-Methylation Arrays
Genomic DNA may be isolated using customer preferred methods. Intact genomic DNA should form a band on a 1.2% agarose gel > 10 kb. If a smear is present the DNA has degraded and may not be suitable for processing.
Sample Preparation for NimbleGen Gene Expression Arrays
User is responsible for doing their cDNA synthesis reactions. cDNA synthesis can be achieved by using Invitrogen’s Superscript Double-Stranded cDNA Synthesis Kit.
Labeling and Hybridization
We are currently using both direct and indirect labeling methods for preparation of targets for hybridization to the arrays. Use the following links to view the details of each method.
Direct Labeling Methods
Indirect Labeling Methods
Affymetrix Probe Arrays
Contact the Genomics Core Facility for protocols for target preparation using dIVT method and for target preparation for SNP genotyping arrays.
GeneChip Eukaryotic Small Sample Target Labeling
The Genomics Core Facility provides three types of Affymetrix platform Gene Expression services:
- Complete Service
- Single in Vitro Transcription (IVT)
- Double in Vitro Transcription (dIVT)
GeneChip Eukaryotic Small Sample Target Labeling describes in detail the methods used.