Affymetrix Guidelines & Preparation

Affymetrix Guidelines

As part of our service fee, all incoming samples are initially checked for quality (Agilent Bioanalyzer 2100) and quantity/concentration levels (NanoDrop ND-1000 spectrophotometer) prior to initiation of any processing steps.

3'-Biased Expression Arrays

We follow prescribed Affymetrix protocols as listed in the GeneChip Expression Analysis Technical Manual in processing all provided total RNA samples destined for analysis on Affymetrix’s 3’-biased expression arrays.

Exon/Whole Transcript Expression Arrays

As with the samples destined for 3’-Biased Expression array analysis, all incoming total RNA samples to be run out on an Exon Array are initially checked for quality (Agilent Bioanalyzer 2100) and quantity/concentration levels (NanoDrop ND-1000 spectrophotometer) prior to initiation of any processing steps.

When running Exon Arrays, we follow Affymetrix’s protocol as outlined in their GeneChip Whole Transcript (WT) Sense Target Labeling Assay Manual.

Cytoscan HD Arrays

 

Affymetrix Sample Preparation

RNA Isolation

We recommend Qiagen columns for RNA isolation.  We recommend against Trizol purification since any residual phenol may inhibit subequent enzymatic reactions.

Qiagen RNAEasy

Qiagen RNAse-Free DNase Set (For removal of genomic DNA from RNA preps)

RNA Quality

- Free of DNA, salt, phenol and other contaminants that could interfere with enzymatic reactions
- A260/A280 ratio between 1.9-2.1 measured in 10 mm Tris pH 7.6
- Agilent 2100 Bioanalyzer shows no degradation of RNA. RIN 7.0 or above.

RNA Quantity

- 2-5 µg Eukaryotic total RNA for Regular Sample Prep (3' biased expression arrays)

- 500 ng total RNA for Gene 1.0 ST Arrays (Human, Mouse and Rat only) (whole transcript arrays)

Biological or Technical Replicates?

Affymetrix Arrays are technically quite reproducible (0.99) so the expense does not usually justify technical reps.   Biological replicates give much more information about your system.