Systematic Screening of Gene Expression Using a cDNA Macroarray - Drugs of Abuse: Neurological Reviews and Protocols - page 251

1. Thaw the samples.

2. Add one tenth of the total volume of denaturing buffer into the samples and
incubate in a 68°C water bath for 20 min.

3. Add each volume of neutralizing buffer and 5 µ L of Cot-DNA (provided by the

manufacturer) and incubate for 10 min.

4. Add probes to appropriate labeled bottles and hybridize overnight in an incuba-
tion oven.

3.7. Washing and Phosphorimaging
Wash solutions 1 and 2 should be kept at 68°C in a water bath for the
duration of wash.

1. Remove the tubes from the oven and empty the contents into a radioactive
container.

2. Add 50 mL of wash solution 1, shake quickly, and empty into the same container.
Add 50 mL again and incubate at 68°C in the oven for 30 min. Repeat two more
times with wash solution 1.

3. Wash two more times with the more stringent wash solution 2. A fi nal wash in 2X
SSC is at room temperature and occurs in a container on a desktop shaker. This is
a 15-min wash followed by mounting onto laminate plastic.

4. Remove the membranes from the shaker and allow one membrane at a time to
drip dry quickly (about 30 s). Place the membrane on the mounting surface with
the probe surface facing upward. Cover the mounting surface with plastic wrap,
making sure to remove any air bubbles and seal with a thermal bag sealer.

5. Mount the sealed membranes into a phosphorimaging cassette and exposed to
a phosphorimaging plate for 3-5 d, depending on the amount of radioactivity
present.

6. Read and analyze the plate using an imaging program such as Clotech's Atlas
Image 2.01.

3.8. Data Analysis
Data analysis is a critical component of hybridization array experiments
and poses a number of challenges owing to the large amount of data generated
even in a single experiment. The most basic goal of array data analysis is to
identify genes that are differentially regulated. Hybridization arrays are prone
to yielding false-positive results and therefore analysis strategies attempt to
decrease this error rate without increasing false-negatives. The analysis method
described here is a simple, empirical approach. There are now more advanced
statistical approaches to data analysis (9) .