Agilent Technologies Gene Expression FFPE Workflow Quick Start Guide introduces how to use the Agilent gene expression microarray workflow to analyze FFPE samples
This document provides a step-by-step workflow for analyzing total RNA from formalin-fixed, paraffin embedded (FFPE) samples using the Agilent Gene Expression FFPE Workflow. The process involves isolating RNA, preparing a Whole Transcriptome Amplification (WTA) library, amplifying it, purifying the products, and labeling them with ULS for hybridization to microarrays.
The guide lists required equipment such as an Agilent Microarray Scanner, Hybridization Chamber, and Thermocycler. It also specifies necessary reagents including the TransPlex Whole Transcriptome Amplification kit (Sigma), Absolutely RNA FFPE Kit (Stratagene), and various Agilent Gene Expression kits for hybridization and washing.
| Hybridization temperature | 65°C |
|---|---|
| RNA extraction incubation time (Proteinase K Digestion) | 18 hours at 55°C |
| WTA Library incubation step 2 | 70°C for 5 minutes |
| WTA Amplification cycles | 94°C for 20 seconds, 65°C for 5 minutes, for 22 cycles |
| Centrifuge speed for purification | 13,000 rpm (approximately 17,900 x g) |
| Hybridization time and speed | 65°C for 17 hours at 20 RPM |
The protocol recommends incubating the tubes at 55°C for 18 hours instead of the standard 3 hours.
The protocol uses the TransPlex Whole Transcriptome Amplification kit from Sigma, specifically part number WTA1-50RXN.