Affymetrix Guidelines & Preparation

Affymetrix Guidelines

As part of our service fee, all incoming total RNA samples are initially checked for quality (Agilent Bioanalyzer 2100) and quantity/concentration levels (NanoDrop ND-1000 spectrophotometer) prior to initiation of any processing steps. If any of the provided samples fail either of these two initial checks, work will be halted and the user will be contacted.

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. Depending on the amount of supplied sample materials, either one or the other of two routinely utilized in-vitro transcription (IVT) protocols is performed:

A. Standard Protocol – Single Round of Amplification

We typically request that users provide us with 11 ug of total RNA/sample (at a concentration of 1.25 ug/uL) when running the standard protocol. This individual sample amount allows for more than adequate levels for sample quality assessment (QC on Agilent Bioanalyzer 2100), sample quantification (on NanoDrop ND-1000 spectrophotometer) and very robust sample processing using the standard protocol. However, we recognize that sometimes due to the experimental design, size and/or difficulty in isolating some specific tissues, you might only be able to provide as little as 1.5 to 2.5 ug of total RNA/sample. In most cases, assuming you have very good quality RNAs, this amount of starting material should prove to be sufficient to run the standard protocol and still obtain the required amount of biotinylated-cRNA required for subsequent fragmentation and hybridization steps.

B. “Small Sample” Protocol Approach

For users whose experimental design (such as total RNA isolated from tissue biopsies or laser capture micro-dissections) only allows for the isolation of much less material than the micro-gram quantities normally required to perform the above listed standard approach, we offer small sample preparation, processing and labeling using Nugen Technologies' Ovation SPIA Amplification and Labeling System. When starting with nano-gram amounts of total RNA and using the Ovation protocol, we are able to readily obtain the necessary amount of DNA required for subsequent fragmentation, labeling and hybridization onto GeneChip arrays. Please check with the Facility staff concerning the minimum amount of starting total RNA material required when contemplating utilizing this approach.

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. If any of the provided samples fail either of these two initial checks, work will be halted and the user will be contacted.

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

Tiling Arrays

The GeneChip Whole Transcript (WT) Amplified Double-Stranded Target Assay Manual outlines a protocol designed to generate double-stranded labeled DNA targets from the entire expressed genome. Tiling arrays offer the ability to carry out unbiased genome-wide transcript mapping studies. This protocol, which we follow, has been designed for use with either the Human Tiling 1.0R Array Set, Mouse Tiling 1.1R Array Set or other species tiling array sets for RNA mapping applications in order to probe expression across all regions of the genome. The procedure described in this Manual recommends as little as 3 µg of total RNA or 300 ng of poly-A+ RNA as starting material from human or mouse samples (actually a little more than indicated here required to allow for upfront QC testing purposes). And because the quality of the RNA is essential to the overall success of the analysis, when using total RNA as the starting material it is checked prior to the rRNA depletion step on an Agilent Bioanalyzer. Both the amplified and un-amplified versions of the assay produce double-stranded, labeled DNA target, which interrogate the genome for all regions of expression. The specific protocol used is dependent on the sample and array type chosen.

Chromatin ImmunoPrecipitation (ChIP) Assays

These tiling arrays along with the newer Human and Mouse Promoter 1.0R Tiling arrays can also be readily used for Chromatin ImmunoPrecipitation (ChIP) assays (sometimes referred to as ChIP-on-chip studies) in order to study transcription factor binding sites, histone protein modifications and other chromatin-protein interactions. For details please see the Affymetrix Chromatin Immunoprecipitation Assay Protocol Manual.

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.