Bauer Core Standard Protocol
Title: Fragmentation of cRNA for Affymetrix GeneChips
Pages: 2
Revision:
Date: August 26, 2003
Author(s): Jennifer A. Couget
Reviewers: Shufen Meng
Contact: affyinfo@cgr.harvard.edu
Comment:
1. Purpose
This protocol describes the fragmentation procedure of cRNA for Target Cocktail
Hybridization of Affymetrix GeneChips.
2. Materials
5X Fragmentation Buffer (stored at room temperature)
DEPC-water
Labeled cRNA
0.65 ml RNase-free tubes to prevent evaporation
3. Instrumentation
To maintain proper fragmentation temperature, using a Thermal Cycler is recommended,
but not required.
4. Reagent preparation
5X Fragmentation Buffer may be bought as part of the Affymetrix GeneChip Clean-up
Module (Affymetrix, p/n 900371)
5X Fragmentation Buffer
200mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc
1M Tris acetate pH 8.1
MgOAc
KOAc
DEPC-water
4 ml
0.64 g
0.98 g
to 20 ml
Mix thoroughly and filter through a 0.2 m vacuum filter. Aliquot and store at room
temperature.
5. Procedure
5.1
It is usually best to perform the fragmentation on the same day as hybridization
whenever possible to minimize freeze-thawing effects on cRNA.
5.2
Quantify cRNA concentration and A260/A280 ratio. cRNA must be at a minimum
concentration of 0.6 µg/µl. If it is not, concentrate under vacuum without heat.
A260/A280 ratio should be between 1.9 and 2.1.
5.3
Use 2 µl of 5X Fragmentation Buffer for every 8 µl of cRNA plus water together
(ratio is 2:8). In addition, the cRNA final concentration in the fragmentation
reaction must be no less than 0.5 µg/µl.
5.4
We recommend fragmenting 20 µg of cRNA in a 40-µl fragmentation reaction.
5.5
For example, cRNA with a concentration of 1 µg/µl:
cRNA (1 µg/µl)
DEPC-water
5X Fragmentation Buffer
Total Volume
20 µl
12 µl
8 µl
40 µl
Final concentration 0.5µg/µl
Final concentration 1X
5X Fragmentation Buffer 8 µl: cRNA plus water 32µl
ratio is 2:8
5.6
Incubate at 94C for 35 minutes. Cool to 4C and place on ice.
5.7
Check quality of cRNA by Agilent Bioanalyzer using mRNA Smear Nano Assay.
5.8
Run 100 ng of unfragmented cRNA side-by-side with 1 µl fragmented undiluted
cRNA directly from fragmentation reaction.
5.9
Assay should reveal long unfragmented cRNAs, majority greater than 400 bp and
fragmented cRNAs ideally between 35 and 200 bp.
5.10
Use all remaining fragmented cRNA in target hybridization cocktail immediately.
5.11
Store fragmented cRNA at –20C short-term for immediate future hybridization.
5.12
Store unfragmented cRNA and extra fragmentation reactions at -80C long-term.