Experimental Protocols

Note: All procedures are done at room temperature and with double distilled water unless otherwise stated.

NEW protocols - June 2007

Post-Processing of Oligo Arrays on Corning Ultra GAPS slides

Post-Processing of Oligo Arrays on PolyLysine coated slides

Ambion MessageAmp II Amino Allyl and Cy Dye Coupling for Oligo Arrays (PDF Version) (updated 05/18/2006)

Preparation of Fluorescent DNA Probe from Total mRNA - Direct label of Total RNA for Oligo Arrays (PDF version) (updated 05/18/2006)

Array Hybridization and Washing for Oligo Arrays (PDF version)

Reverse Transcription (RT) and Amino-Allyl coupling of cDNA for (total) RNA sample input

Cy5 destruction has been reported after 10-30 seconds exposure to ozone levels above 5-10 ppb, especially during the array washing, drying and scanning steps. We have analyzed this problem and developed several solutions you can download here (updated June 2007):

Details on ozone destruction of Cy5 and plans for building excellent ozoone-free enclosurese.

A Good Cheap Kludge - how to build an ozone-free room.

How to dry microarray slides with an ozone-free room and small centrifuge.


DNA Preparation
Preparation of DNA Samples (updated 12/3/1999)
Slide Preparation
Poly-l-Lysine coating of slides (updated 10/6/1999)


NOTE: The following are not recommended and are replaced by the above protocols.

RNA Amplification
aRNA Amplification Ambion method (updated 1/28/2003)
aRNA Amplification Wang protocol (updated 4/23/2002)
aRNA Amplification (updated 6/15/2001)

Nucleic Acid Preparation

Human FastTrack mRNA Isolation (updated 8/9/1999)

Preparation of total RNA from whole blood collected in PAXgene tubes (updated 6/18/2002)

Nucleic Acid Labeling
Reverse Transcription using Cy3- & Cy5-dUTP:
---Yeast (updated 3/29/1999)
---Human (updated 3/15/2001)
---E. coli (updated 2/22/1999)
Reverse Transcription and aa-UTP Labeling of RNA (updated 6/28/2000)
Revised amino-allyl labeling protocol (updated 2/3/2001)
Labeling Genomic DNA (updated 11/17/2000)
Post Processing
Post-Processing of Arrays (updated 9/19/1999)
Array Hybridization-General/Yeast (updated 8/10/1999)
Array Hybridization-Human (updated 3/15/2001)
Cy dye HPLC purification
Design of Yeast Genomic Expression Experiments

Post questions on the Microarray Forum.