Expression Microarray Preparation
A oligo-nucleotide microarray hybridization technique was used to identify the novel
genes and described elsewhere [26]. In brief, total RNA was extracted by Trizol®
Reagent (Invitrogen, USA), and followed by RNeasy Mini Kit (Qiagen, Germany).
RNA purified is quantified by OD260nm by a ND-1000 spectrophotometer
(Nanodrop Technology, USA) and qualitated by Bioanalyzer 2100 (Agilent
Technology, USA). 0.5 g of total RNA was amplified by a low RNA input fluor
linear amp kit (Agilent Technologies, USA) and labeled with Cy3 or Cy5 (CyDye,
PerkinElmer, USA) during the in vitro transcription process. Tumor RNA was labeled
by Cy5 and RNA from Universal Human Reference RNA was labeled by Cy3. 2g of
Cy-labled cRNA was fragmented to an average size of about 50-100 nucleotides by
incubation with fragmentation buffer at 60oC for 30 minutes. Correspondingly
fragmented labeled cRNA is then pooled and hybridized to Human 1A (version 2)
oligo microarray (Agilent Technologies, USA) at 60°C for 17 h. After washing and
drying by nitrogen gun blowing, microarrays are scanned with an Agilent microarray
scanner (Agilent Technologies, USA) at 535 nm for Cy3 and 625 nm for Cy5.
Scanned images are analyzed by Feature extraction software (Agilent Technologies,
USA), an image analysis and normalization software is used to quantify signal and
background intensity for each feature, substantially normalized the data by
rank-consistency-filtering LOWESS method.
Real-time PCR Quantification
Relative cDNA quantitations for OPN and an internal reference gene (GAPDH) were
done using a fluorescence-based real-time detection method by LightCycler Faststart
DNA Master SYBR Green I [Roche Diagnostics GmbH, Roche Applied Science,
Germany] as described previously [27-29]. The PCR reaction mixture consisted of 0.5
μM each of the primers (OPN: forward 5’-CGAGGAGTTGAATGGTGCATAC-3’;
reverse 5’-TTTCAGCACTCTGGTCATCCA-3’; GAPDH: forward
5’-TGCACCACCAACTGCTTAGC-3’; reverse
5’-GGCATGGACTGTGGTCATGAG-3’); 200 nM probe; 0.03 unit/μl AmpliTaq
Gold Polymerase; 200 μM dNTP; 3.5 mM MgCl2; and 1× TaqMan Buffer A, which
contains a reference dye, to a final volume of 25 μl (all reagents were from
Perkin-Elmer Applied Biosystems). Cycling conditions were 95°C for 10 min,
followed by 45 cycles at 95°C for 10 s and 55°C for 5 s and extend at 72°C for 10 s.
For each sample, parallel real-time PCR reactions were performed for the gene of
interest and the GAPDH reference gene to normalize for the input of cDNA. The ratio
between the values obtained provided relative gene expression levels for the gene
locus investigated. Among the 58 pairs of studied specimens (tumor and normal
tissues), 14 of them were repeated to measure OPN mRNA levels in tumor and
normal tissues by real-time PCR. We found the high correlation of tumor-to-normal
ratio of OPN mRNA levels between the first and second time measurement
(Spearman correlation coefficient r = 0.99, n = 14, p < 0.0001), suggesting good
laboratory quality control.