patient identifier: 24 disease: clear cell renal cell carcinoma (RCC) tissue: adjacent nontumor kidney tissue gender: male age at surgery (yrs): 75 patient status: cancer-specific death
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified from tissue extracts by the Trizol protocol (Technologies, Rockville, MD). Total RNA was treated with RNase-free DNase (RNeasy - Qiagen) for 15min to minimize genomic DNA contaminants. Purity of the isolated RNA was estimated by measuring the ratio A 260/A 280. Integrity of the total RNA was checked by electrophoresis using the total RNA Nano assay in the 2100 Bioanalyzer (Agilent Technologies).
Label
Cy5
Label protocol
Complementary RNA (cRNA) was obtained by amplification following the Wang protocol method (Wang et al. Nat Biotechnol 2000, 18:457-459). Briefly, using a poly-dT primer incorporating a T7 promoter, cDNA was synthesized from 3 ug total RNA using Superscript III enzyme (Invitrogen, Carlsbad, CA). Double-stranded cDNA was obtained using a template switch oligo primer and the Advantage cDNA Polymerase mix kit (Clontech Laboratories, Mountain View, CA). Double-stranded cDNA was purified by phenol/chloroform extraction. The aqueous phase was isolated using Phase-Lock Gel Heavy (Brinkmann Instruments, Westbury, NY) and cDNA was precipitated with ethanol. Subsequently, cRNA was generated from the double-stranded cDNA template through in vitro transcription with RNA Polymerase T7 MegaScript (Ambion, Austin, TX). The amplified cRNA was purified using TRIzol reagent (Life Technologies). A second round of amplification was performed with 1 ug of cRNA obtained in the previous step, in the presence of amino-allyl-UTP (Ambion). Amino-allyl-cRNA was treated with RNase-free DNase (RNeasy Mini kit; Qiagen) to remove potential DNA contamination, quantified by spectrophotometry (Nanodrop), and checked for quality with a Bioanalyzer 2100 system (Agilent). A coupling and quenching reaction of amino-allyl-cRNA with Cy5 monoreactive dye was performed (Amersham Pharmacia Biotech, Piscataway, NJ,). Labeled cRNAs were purified from free dye using the RNeasy Mini kit (Qiagen), concentrated with microcon YM-30 columns (Millipore Corporation, Billerica, MA) and resuspended in 50uL of water.
Hybridization protocol
Labeled targets were resuspended in 200 µl of 1 x hybridization buffer (50% formamide, 25% of proprietary Microarray Hybridization Buffer Version 2 from Amersham Biosciences cat. RPK0325), denatured for 2 min at 92 ºC and centrifuged at 13,000 rpm for 5 min. All slide processing steps (blocking, hybridization, washing) were carried out on an automated slide processor (ASP) from Amersham Biosciences. The slides were incubated for 16h at 42ºC and subsequently washed at room temperature in 1xSSC/0.2%SDS for 5min, 0.1xSSC/0.2%SDS for 5 min and in 0.1XSSC for 3 min. After the washing steps, slides were flushed with isopropanol and dried with an air flush at 42ºC.
Scan protocol
Images were obtained by laser scanning using the GenIII Array Scanner (Amersham Biosciences). Slides were scanned with 600 volts of PMT voltage.
Description
24N_rep_L
A total of 3055 spots were considered valid and expressed for analysis from the total of 4608 contained in the platform GPL3985. The Sample data table contains only these 3055 valid spots. Therefore, a total of 1253 spots corresponding to positive controls, negative controls, empty spots or contaminants were eliminated (see platform GPL3985 for a more detailed description). Additionally, 300 spots detected in less than 90% of the arrays in one of the two groups (nontumor or tumor) were filtered. Each array contains one technical replica, named replica left (L) and replica right (R) for each sample.
Data processing
We employed in our analyses the background subtracted Median-based Trimmed Mean (sMTM) density value calculated for each spot by ArrayVision software. The MTM value represents the mean of all the pixels remaining in a target, after first removing pixels with density values that exceed four median absolute deviations (MADs) above or below the median. The MTM density for each spot was subtracted by the median background surrounding the spot (distance of 2 pixels with 3 widths of pixels). Genes were considered expressed if its probe intensity was higher than the local background intensity and above the threshold defined by the average intensity plus three standard deviations of a set of plant-derived negative control cDNA probes (GE Healthcare). Probes were excluded from further analyses when they were detected in less than 90% of the arrays in any of the two groups, nontumor or tumor for the statistical analysis. We normalized expression data (3055 valid spots) between experiments by quantile (Bolstad et al., 2003. Bioinformatics, 19(2): p. 185-93).
Expression analysis and in silico characterization of intronic long noncoding RNAs in renal cell carcinoma: emerging functional associations (RCC malignancy)
