Microarray analysis requires RNA of very high integrity. As a standard practice all samples submitted to the core must be analyzed for integrity using an Agilent bioanalyzer, to confirm that the ratio of 18S:28S ribosomal subunit RNA is close to the theoretical maximum of 2.5. The bioanalyzer provides a complete RNA profile with as little as 5 ng ml -1 total or poly A + RNA and can quickly reveal sample degradation.

 

Your samples can be submitted to us for RNA extraction. This simply involves isolating your sample or cells and lysing them in a denaturing buffer, prior to submitting them. If prepared correctly, almost all RNA samples prepared from animal tissues should look like the high quality RNA trace shown to the left. However, with some tissues that have a high RNase content, such as adult pancreas, a trace similar to that of the partially degraded RNA sample might be expected. It is very important that there is consistency in the quality of your RNA samples within an experiment, and samples that produce a high quality trace should never be compared with a sample that showed partial degradation. At our discretion we will reject samples that we consider to be of low quality, as our experience has shown us that they will not produce reliable data.

RNA Amplification

Typically our microarray hybridization experiments require 10-20 m g of total RNA. However, many investigators are performing experiments using small amounts of material (e.g. sorted cells, laser capture or islets). We can successfully amplify RNA from as little as 5-25 ng of total RNA (1000 cells or 1 islets). We use Nugen’s Ovation™ Aminoallyl RNA Amplification and Labeling System which involves a series of enzymatic reactions resulting in linear amplification of exceedingly small amounts of RNA for use in array analysis. Unlike exponential RNA amplification methods, such as NASBA and RT-PCR, Ovation amplification maintains representation of the starting mRNA population.

Islet Purity Matching

Our extensive experience in working with islet samples has enabled us to develop an Islet Purity Matching assay that we have found to be essential for the production of high quality microarray analysis from islets. In outline, QPCR is used to determine the relative levels of Insulin and Amylase in each sample. This then allows us to only hybridize samples that are well matched for purity in terms of the amount of exocrine contamination found in every islet preparation. In this way differential gene expression as a result of the islet preparation technique can be removed, allowing the investigator to identify to accurately determine islet gene expression profiles.  

Sample Labeling & Hybridization

Expression Analysis . From as little as 100 ng of total RNA (or 20 m g without amplification) our low cost fee for expression analysis includes:

• Indirect amino-allyl labeling of your sample, using methods developed in-house that significantly enhance the sensitivity & accuracy of the final result.
  

• Hybridization and scanning using SureScan technology with Agilent Technologies automated, dual-laser microarray scanner. Its dynamic autofocus technique constantly adjusts the focus of the scanner to compensate for glass-warp and curvature found in most glass slides. This delivers vast improvements in sensitivity and overall confidence in scanned results.

• Image analysis, data normalization and statistical identification of differentially expressed genes. Using our high level experience in microarray analysis we will work with the end user to provide a list of only those genes that were differentially expressed as determined using False Discovery Rate statistical analysis and limits on fold change requested by the end user. All clients receive a detailed report on what methodological and statistical analysis was performed, and a final results table is provided in easy to use Microsoft Excel format. We also have options for advanced analysis.

Promoter Analysis. From 1 m g of amplified DNA we will label, hybridize, scan and perform data analysis similar to the above. See the description of the Promoter Chip BCBC-5A.0 for more details.

Advanced Analysis

In addition to the basic analysis provided in our analysis report, we provide several options for advanced analysis to help the investigator determine how best to interpret the results and to prepare the data for publication. Identification of biological pathways in a set of differentially expressed genes using programs such as Ingenuity or GenMapp can be particularly useful when dealing with a large set of differentially expressed genes. Identification of functional overrepresentation (EASE analysis) and clustering analysis are other methods that we commonly use.