New Molecular Approaches to Tissue Analysis

Tissue Sampling

To maximize the recovery of RNA and protein, transrectal biopsies and samples removed from radical prostatectomy were snap-frozen in liquid nitrogen cooled isopentane (Scott et al. 2000). Hematoxylin- and eosin-stained sections cut on a cryostat and mounted on charged glass slides were used for microdissection using the PixCell Laser Capture Microdissection Microscope (Arcturus Engineering; Mountain View, CA) essentially as described by Emmert-Buck et al. (1996). Total RNA was extracted from laser captured cells using the Micro RNA Isolation Kit (Stratagene; San Diego, CA) (Luo et al. 1999).

RNA Amplification and Reverse Northern Blotting Analysis

The purified RNA sample underwent amplification using 0.5 μg/μl T7-oligo dT primer, Superscript II Reverse Transcriptase (Life Technologies; Huntsville, AL), and Ampliscribe T7 Transcription Kit (Epicentre Technologies; Madison, WI) as previously described (Luo et al. 1999). Ten μl of purified, resultant amplified RNA (aRNA) underwent second-round amplification using pdN6 random hexamers (1 μg/μl; Pharmacia, Piscataway, NJ) during first-strand synthesis and T7-oligo dT primer for the second-strand reaction, as previously described (Luo et al. 1999).

Second-round aRNA specimens were synthesized into randomly labeled α[³²P]-dCTP probes using pdN6 random hexamers (1 μg/μl; Pharmacia) and the Superscript PreAmplification System reagents (Life Technologies) as previously described (Poirier et al. 1997). About 0.5 μg of target cDNA was crosslinked to the blot membrane with a UV Stratalinker 2400 (Stratagene). The positive control for each blot consisted of 0.5 μg of GAPDH. The negative control was a single-strand antisense laminin 5 β3 cDNA. Reverse Northern blots were carried out as previously described (Poirier et al. 1997). In brief, 500 ng of target cDNAs including controls were denatured, combined with 111 μl sterile water, 80 μl 1 M NaOH, and 4 μl 0.5M EDTA, and crosslinked to an Ambion Bright Star Plus positively charged nylon membrane (Ambion; Austin, TX) using a Minifold II slot-blot apparatus (Schleicher & Schuell; Keene, NH). The membranes were prehybridized at 68C for 45 min in Perfect Hyb Plus solution (Sigma; St Louis, MO). Radiolabeled cDNA probe was denatured at 100C for 10 min, added to the prehybridization mixture, and hybridized overnight at 68C in Perfect Hyb Plus solution. The membrane was washed twice for 20 min each in 2 × SSC/0.1% SDS at 68C for 15 min. Higher-stringency washes were performed in sequence as necessary to remove background signals from the membrane, including two 0.2 × SSC/0.1% SDS washes at 68C for 15 min and two 0.1 × SSC/0.1% SDS washes at 68C for 15 min. The blots were then exposed on phosphor imaging screens (Molecular Dynamics; Sunnyvale, CA). Each blot was measured and plotted with a Phosphorimage 445SI scanner (Molecular Dynamics). Blots were stripped and re-probed up to four times.

Microarray Analysis

cDNA arrays were constructed on glass slides adapting the approach pioneered by Brown and colleagues (Schena et al. 1995; DeRisi et al. 1996; Brown and Botstein 1999). PCR-amplified cDNAs obtained from Research Genetics were spotted onto chemically activated glass slides in defined arrays using a OmniGrid DNA robotic Printer (Gene Machine, Genomic Instrument Services; San Carlos, CA) equipped with Telechem print heads and quills. Two type of arrays were utilized: (Scott et al. 2000) a 576-gene array comprised of 544 cancer-related genes, 24 housekeeping genes, and 8 ice plant genes used as controls (Emmert-Buck et al. 1996). A 5700-gene array was composed of 5184 genes and ESTs released as the first gene set by Research Genetic (gf200) plus an additional 516 genes collected from various research labs within the Arizona Cancer Center. Approximately 5 μg of mRNA isolated from cells or amplified from tissue microdissection was used for gene analysis. Firststrand cDNA was made in the presence of Cy5-dCTP (red) or Cy3-dCPP (green). The two fluorescent first-strand cDNAs were mixed, denatured, and used as targets for the genes on the microarray slides. After hybridization and wash steps, emissions were collected using a Scan Array 3000 microarray reader (General Scanning, Packard Biochip Technologies; Billerica, MA) and quantitated using the Imgene 2.0 software (Biodiscovery; Los Angeles, CA).