RNA Expression: CodeLink
Other RNA Services
The CBF utilizes the Ambion MessageAmp Premier labeling assay to generate hybridization targets for CodeLink microarray studies. Investigators may choose to have the CBF extract and prepare RNA for them directly from frozen tissues or cell samples, or they can choose to submit investigator-prepared RNA.
High quality RNA is critical to the success of a microarray expression assay, so all samples generated within the CBF and investigator-prepared samples will be assessed for quality via the Nanodrop spectrophotometer and the Agilent Bioanalyzer. Investigators will be notified of all samples that do not meet Quality Control criteria for quantity and quality. Please see Sample Requirements below for more information.
CodeLink whole genome expression arrays are available for human, mouse, and rat.
Human Whole Genome Array - comprehensive coverage of the human genome, targeting ~57,000 transcripts and ESTs (expression tagged sequences).
|CodeLink™ Human Whole Genome Probe-Target Classification|
|Major biological process||Number of genes present|
|Transcription and translation||~2,700|
|Immune and inflammation response||~1,100|
Mouse Whole Genome Array
|Number of probes||35 587 (Total) 34 967 (Unique sequences)|
|Number of transcripts||36 142|
|Number of probes targeting transcripts other than controls||34 967|
Rat Whole Genome Array
interrogate approximately 34,000 transcripts representing most of the known and predictive genes of the rat genome, as it is described today in the public domain.
|Number of probes||33 849|
|Number of transcripts||32 590|
|Number of probes targeting transcripts other than controls||29 842 (Based on unique UniGene IDs)|
|Expression Array Type||Target Prep Method||*^Minimum Amount of Total RNA Required||Minimum Concentration|
|3' IVT||Ambion MessageAmp Premier Kit||500ng||166.67ng/ul|
|*Quality, Quantity, and Concentration subject to verification by the CBF|
|^ These are the minimal amounts specified in the respective protocols. We recommend you provide us with excess in order to run QC analysis and to repeat assays if necessary|
High quality array data depends upon high quality samples!
RNA: Prior to target synthesis, the CBF will evaluate all submitted RNA samples for quantity and quality (amount of degradation). RNA will be quantified by UV absorbance using a Nanodrop spectrophotometer and validated to be non-degraded via the Agilent Bioanalyzer. A260:A280 ratios should be 1.8-2.0 and the RIN should be >7.0. Submit RNA in nuclease-free water.
Tissue or cells may also be submitted to the CBF for extraction prior to performing an expression array assay. For researchers who are collecting tissue on their own, we suggest a few guidelines to limit RNA degradation and ensure sample quality. All samples submitted to the lab for extraction and downstream microarray applications should be handled in one of the following ways at the time of collection:
- Fresh tissue should be flash frozen in 2.0 mL or Nunc tubes in LN2 or dry-ice and maintained at -80°C until submitted to the CGIF. Cells should be pelleted in 1.5-2.0 mL tubes, all supernatant removed, and then flash frozen in LN2 and maintained at -80°C until submitted to the CBF.
Tissue and cell pellets should be placed in RNAlater (Ambion, Inc) and stored at 4°C for up to one month, or
Tissue and cell pellets should be placed in RNAlater at 4°C overnight, then stored at -20°C indefinitely until submitted to the CBF, or
Tissue should be placed in RNAlater at 4°C overnight, then removed from the RNAlater and stored at -80°C indefinitely until submitted to the CBF. Cell pellets should be placed in RNAlater at 4°C overnight, then stored at -80°C indefinitely until submitted to the CBF.
- Tissue and cell pellets should be homogenized immediately in either Qiagen Buffer RLT (recommended) or Trizol (not recommended) and then stored at -80°C until submitted to the CBF.
Once samples have been extracted they will be assessed for quality, quantity, and concentration prior to beginning microarray assays.
Investigators will be notified if a sample is of insufficient quantity or quality (failed Quality Control parameters) for optimal array performance.
Samples cannot be processed until a completed Service Request Form is received along with a signed estimate. Click here for pricing information.
*Please note that CodeLink arrays are only available in 6 packs and investigators will be billed for any unused arrays.
Technology and Equipment
Genepix 4000B Microarray Scanner
The GenePix 4000B Microarray Scanner acquires data at two wavelengths simultaneously. This means drastically reduced scan times and hence greater workflow efficiency. Apart from superior speed, simultaneous scanning eliminates potential errors associated with aligning two separately scanned images, and provides real-time access to data as they are acquired.
With automated photomultiplier (PMT) balancing, the fully integrated GenePix Pro Acquisition and Analysis Software controls the GenePix 4000B Microarray Scanner and can automatically evaluate the required PMT gain values, for fast and easy optimization of signal intensity and channel balance. The GenePix 4000B Microarray Scanner then acquires data at your selected resolution between 5-100 micron, allowing the adjustment of image resolution and file size for each experiment.