Target Discovery and Validation Reviews and Protocols

Gene Target Discovery 125
Fig. 1. Processing steps in rescue suppression subtractive hybridization (SSH). First,
SSH was carried out using the PCR-selected cDNA subtraction kit (Clontech). PolyA+
RNA (2 µg) was reverse transcribed according to manufacturer’s instructions and then
digested with RsaI restriction enzyme. The adapters were ligated to the tester cDNAs and
the ligation efficiency was investigated. Two rounds of hybridization with an excess of
driver cDNA and one PCR amplification were performed with the driver cDNA.
Subsequently, two rounds of PCR were performed to obtain differentially expressed genes
from which a radioactive probe was prepared. Second, total RNA (20 µg/lane) from either
the tester or the driver was electrophoresed on 1% agarose/formaldehyde gels, transferred
to Hybond N+ membrane, and UV-cross-linked. Membranes were hybridized with
32 P-labelled probe representing the entire subtracted PCR fragments, which are expected
to contain genes overexpressed in SKBR3 (tester, left), but not in HMEC cells (driver,
right). Third, the nitrocellulose portion containing differential expressed genes were
marked, cut out, and incubated in 100 µL of water for 10 min at 95°C. Eluted probe molecules
(10 µL) were reamplified in a 50-µL PCR reaction by using the secondary PCR conditions
as described in the PCR-selected cDNA subtraction kit. The PCR products were
resolved on 2% agarose gels and then purified using the QIAGEN gel extraction kit as
described by the manufacturer. Fourth, the PCR-reamplified probe molecules were cloned
into the pGEM-T vector (Promega) according to the manufacturer’s instructions, and positives
clones were sequenced using either T7 or SP6 primers by automatic sequencing. The
identity of the sequenced clones was identified by Blast searches. Fifth, individual gene
probes were used in Northern blots to confirm their differential expression (21).
represented by sequences on the array, i.e. known genes. Significant transcriptional
changes of excluded genes that may be of importance thus are missed.
However, by arraying amplified open reading frames from genomic DNA,
unknown genes can be analyzed for differential gene expression, and alternative