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Microrray

Please contact Robert Hill if you have any questions Robert-Hill

The GeneAtlas microarray system is a complete microarray system for measuring whole-genome expression on Affymetrix manufactured GeneAtlas Array Strips. It utilizes a relatively simple work flow and data analysis is performed in Pathway Studio software customized for this machine. Each array strip houses 4 chips and is thus able to process 4 different samples. The staining and washing of the hybridized probes is performed individually but is done with robotics thus allowing you to safely run more than one Array Strip in an experiment. Our facility currently has a 4-chamber hybridization oven. Thus the maximum number of samples you can run in one experiment is 16 (using 4 Array Strips).

Guide for working with RNA

Guide to analizing your Agilent Bioanalizer Results

Robert Hill

 

Cost

The data acquired from a microarray experiment can be priceless but the cost still significant. The most crucial factor affecting cost in a microarray experiment is experimental design. We will work with you extensively to ensure that your experiment is designed appropriately to minimize cost while maximizing data capture. To calculate the cost of your experiment, combine the cost of the following::

  • Array strips (one strip =4 samples)
  • Prep reagents (IVT or WT) (Enough for your samples)*
  • Staining reagents (Enough for your samples)*
  • Service charges

*Some reagent bundles from the manufacturer will provide more material than needed for your experiment. The core facility will work with you to help that you purchase only those reagents that will be needed in your experiment, but can not guarantee this until a quote is generated for your specific experiment.

Please fell free to contact us for a quote.

Sample Preparation Options & Arrays

  • Affirmative provides two different experimental systems for the 3 more commonly processed arrays (mouse, human and rat)
    • The WT (Whole Transcript) system evaluates the entire mRNA transcript for each gene.
    • The 3'IVT (in vitro Translation) system uses probes that emphasize the 3' region of each transcript.

Method

Background and Description

Compatible GeneAtlas Array Strips

3' in vitro transcription (3' IVT) Expression Analysis

 

The 3' IVT (in vitro Transcription) method of target preparation is compatible with Array Strips that generally focus on hybridizing to the 3' end of the transcript.

This principle of target preparation is based upon linear RNA amplification and employs T7 in vitro transcription, also known as the Eberwine or reverse transcription-IVT (RT-IVT) method. This process is considered the gold standard for target preparation for gene expression analysis. RT-IVT was experimentally validated using TaqMan® RT PCR (MAQC Consortium et. al, 2006).


In this method, total RNA undergoes reverse transcription to synthesize first-strand cDNA. This cDNA is then converted into a double-stranded DNA template for transcription. In vitro transcription synthesizes aRNA and incorporates a biotin-congugated nucleotide (cRNA is also known as amplified RNA or aRNA). The aRNA is then purified to remove unincorporated NTPs, salts, enzymes, and inorganic phosphate. Fragmentation of the biotin-labeled aRNA prepares the sample for hybridization onto the array strip.

3'IVT sample preparation reagents (Chips not included):

10 reactions $980.00
30 reactions $2,880.00

 

 

3' Array Strips

The HG-U219 array utilizes probes that focus on established, well annotated genes. The PM (perfect match) arrays for mouse and rat species provide whole genome coverage of the transcribed mouse and rat genomes in a single array.

 

 

Method

Background and Description

Compatible GeneAtlas Array Strips

Whole Transcript (WT) Expression Analysis & Profiling

The WT (Whole Transcript) method of target preparation generates biotinylated sense-strand DNA targets from the entire expressed genome without bias. The assay and the associated reagents are for use strictly with ST (Sense Target) array strips. The probes on these arrays have been selected to be distributed throughout the entire length of each transcript.

The protocol is based on a novel reverse transcription priming method that eliminates the need for a separate rRNA depletion step. The reagents includes RT primers designed with a proprietary oligonucleotide matching algorithm that eliminates primer sequences with homology to known ribosomal RNA sequences. This results in complete and unbiased coverage of the transcriptome, with a significant reduction in rRNA amplification as compared to other methods.

Each array is comprised of more than 750,000 unique 25-mer oligonucleotide probes that interrogate more than 28,000 genes. Discovery content, such as transcript regions supported by more speculative sources including expressed sequence tags (ESTs) and gene predictions, is not interrogated.

Cost of WT Reagents(cost of strip not included):

10 reactions $ 630.00
30 reactions $1,030.00

ST Array Strips

The ST 1.1 Array Strips are available for the following species:

All ST1.1 strips are $820.00 per strip (4 samples)

 

All Prices are current as of Dec 2012. Please also visit our pricing page

Service Charges

Service Charge
Hybridization, washing and scanning of Array Strip. Generation of expression cell data files $75.00 for 1st Array Strip. $35.00 for each additional (up to 4)
Preparation of hybridization cocktail *$175.00 for 4 samples. *$74.00 for each additional set of 4 samples (up to 16 samples total)

*Excludes cost of reagents

The preparation kits and Array strips can be purchased individually or bundled. Please contact the CMMB Core Facility to get a more accurate cost estimate.

Work Flow

  • It takes three days from sample collection to expression analysis to process one array strip. The 3' IVT system for example outlines the following work flow. Note that the WT preparation method varies significantly in protocol and reagents but there is little difference in time or complexity. If you are going to prepare the sample for hybridization, review the protocols.
  • The CMMB Core Facility will prepare your validated RNA samples for hybridization for you. Fees vary depending on sample number and preparation method. As a general guide, the preparation of 4 samples (excluding reagents) cost an additional $175.00. Scanning of your Array Strips costs $50.00 for one strip + $20.00 for each additional strip (up to 4 strips - 16 samples)
  1. Purification of total RNA
  2. ds-cDNA synthesis
  3. in vitro transcription (IVT)
  4. cRNA biotinylation & purification
  5. cRNA fragmentation
  6. Preparation of the hybridization cocktail
  7. cRNA hybridization to the array strip
  8. Washing/staining of the array strip
  9. Scanning
  10. Analysis

Experimental design

The most important component of running a biologically informative microarray experiment is the initial design of your experiment. The links below can assist you in the design of your experiment.

Microarray Experiments: A conceptual View and Practical Considerations (flash player)

Microarray Experiments: Statistical Tools and Data Visualization (flash player)

Fundamentals in Microarray Data Analysis (pdf)

Microarray experimental design: power and sample size considerations (pdf)

Data Analysis

  • Affymetrix Expression Console (v1.02)

    • Provides signal estimation and QC functionality for Expression Arrays allowing users to:
      • Generate probe set summarization (CHP) files from feature
        intensity (CEL) files for both 3' Expression Arrays and Exon
        Arrays.
      • Capture a standard set of metrics for evaluating the success of the
        individual hybridizations for both 3' Expression Arrays and Exon
        Arrays.
      • Identify outlier samples in the data set.