RNA Preparation and Sample Quality Testing

The Microarray Core has the following guidelines for researchers preparing RNA for microarray experiments:

• High-quality RNA is the most important determinant of successful microarray experiments. The utmost care should be taken to provide pure, nondegraded total RNA samples to the core.
• We have found that RNA prepared with Qiagen column systems works well. Care must be taken, however, to remove all residual wash buffer and ethanol from the column before the final sample elution step. We strongly recommend both spinning the column at full speed for at least two minutes, and then applying the column to a Qiagen vacuum manifold system for five minutes to evaporate the remaining ethanol before eluting the sample. Alternatively, one may spin the column for two minutes, and then move the column to a fresh empty tube and spin an additional three minutes.
• If the RNA is isolated using TRIzol, RNAzol, or a comparable procedure, then we strongly recommend using a Qiagen RNeasy cleanup kit to further purify samples.
• For larger tissue samples we recommend immediately freezing with liquid nitrogen, and then grinding to a powder with a mortar and pestle. The powder can then be placed in a TRIzol reagent or other lysis buffer. Placing chunks of tissue into lysis buffer directly often leads to RNA degradation.
• A DNase treatment is strongly recommended. It removes genomic DNA that may interfere with the experiment.

• The volume of the sample should not exceed 12 ul.
• The OD 260 / 280 ratio should be at least 1.8 for pure RNA.
• Be sure to clearly label sample tubes. Include a distinct name as well as the concentration of each sample for efficient processing.

Target Amplification Protocols

The core offers multiple target amplification protocols for the preparation of biotin-labeled target. The following table indicates which protocols are available based upon one’s preference of array type.  Furthermore, when selecting a protocol one must consider both the quality and quantity of the starting RNA.

Analysis with GeneChip Microarrays

Whole genome expression monitoring will have extraordinary impact on clinical diagnosis and therapy and bring new power to both basic research and clinical medicine. As the field progresses, we will identify new probes for cancer, infectious disease, inherited disease, DNA genetic damage, analysis of gene expression and analysis of protein expression. Equally important will be new therapeutic tools in the form of recombinant gene products, novel drug targets, rational drug design and gene therapy. Next-generation efforts will allow us to link gene expression patterns with formal characteristics of disease models including histological, pathological and clinical state descriptions.
Affymetrix Inc. has pioneered techniques for the photolithographic synthesis of oligonucleotide arrays at very high density on silicon chip surfaces (Pease et al., 1994) . The Affymetrix system can analyze either expressed mRNA or DNA gene sequences (Lipshutz et al., 1999). Recent improvements in the production of new chips with 25-mer versus previous 16-mer oligonucleotides and with much less chip-to-chip and position-to-position variation than was previously observed. The procedure is to purify RNA or DNA from the biological material to be analyzed and subject it to reverse transcription / cRNA transcription or direct PCR, respectively, with the incorporation of fluorescent tags in the final steps of probe labeling. Carefully controlled hybridization and analysis of fluorescent intensities at each position allows abundance estimation with discrimination of specific and nonspecific signals sufficient to detect single base mismatches. The detection of known mutations and alleles, an attractive capability of the Affymetrix system, is applicable to several studies under way here, such as the detection of p53 or BRCA1 mutations or p450 gene alleles that account for slow and rapid drug metabolizers. A particularly powerful application of sequence-specific allelic analysis is to detect resistance alleles in infectious microbial organisms such as tuberculosis and HIV, or the identification of microbial species using conserved regions such as ribosomal genes. A new capability of the Affymetrix system is a multiplex PCR and custom chip technology that is able to “interrogate” 5,200 known regions of the human genome that exhibit single nucleotide polymorphisms (SNPs) (Hacia et al., 1999).
This approach to individual genotyping is likely to be capable of providing excellent data for linkage analysis. Its genome mapping power compared to the approach of using conventional microsatellite markers is not yet clear. However, with so many markers able to be interrogated on a single chip in parallel fashion, it may well prove to be quite practical and cost effective.
In a clinical context, expression profiling using the Affymetrix system will do more than provide a better understanding of biological and pathobiological processes and the possible function of genes and proteins. Tumor types should be exquisitely diagnosed and should lead to optimization of therapeutic approaches. Specific tailoring of therapeutic approaches should be accomplished using expression profiling in individual patients. Structure-function analysis of new drugs may be vastly accelerated, particularly for the optimization of therapeutic index and specificity. This may be accomplishable by application of the GeneChip system to many biological systems including microbial cultures, yeast two-hybrid, cell culture or in vivo models.
A typical experiment would use between two and 20 gene chips for a complete study. The data provided in such an experiment can fuel further research efforts in a laboratory for a year or more.

Array Types and Quality Control Parameters

Affymetrix Exon and Gene Arrays
The Affymetrix Exon and Gene Arrays are the most recently released expression arrays. The Affymetrix Gene Arrays offer complete genome coverage, based on the most recent data. For each gene transcript there are at least 26 unique oligonucleotide probes, whose sequences are scattered across the exons of the entire gene.
The Affymetrix Exon Arrays assay expression of every exon of every gene, allowing global analysis of alternate exon usage. Most investigators use the Affymetrix Gene 1.0 ST Arrays for their projects. Affymetrix has developed a Mouse, a Human and a Rat Gene 1.0 ST Array.
Illumina BeadChip  
The Illumina BeadChip is a commercially manufactured microarray that utilizes BeadArray technology.  These microarrays provide genome-wide gene expression data by using one unique 50-mer gene-specific sequence for each gene transcript.
Affymetrix  3’ Expression Arrays
The Affymetrix  3’ Expression Arrays also offer complete genome coverage for many organisms. For these arrays, gene transcript expression is determined by using 11 unique oligonucleotide probes, whose sequences are located near the 3’ end of the gene transcript.
Remember that all samples must use the same target amplification procedure, and the same microarray type for a single project.
Array Quality Control Parameters
For each sample hybridized to an Affymetrix Expression array, a number of quality controls are checked after the data analysis file is created. An analysis of all quality control parameters indicate whether there was efficient target amplification, labeling and hybridization to the array.
Evaluated expression data is then transferred back to the researcher via a CD or a network drive. Statisticians in the Biomedical Informatics Core are available to provide more in-depth analysis.