|
| Status |
Public on Feb 28, 2017 |
| Title |
Clear cell papillary renal cell carcinoma sample: S_12-36332_Tumor |
| Sample type |
RNA |
| |
|
| Source name |
Hybridization for Clear cell papillary renal cell carcinoma sample: S_12-36332_Tumor
|
| Organism |
Homo sapiens |
| Characteristics |
disease status: Tumor
patient: S_12-36332
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Representative formalin fixed paraffin embedded (FFPE) tissue samples were selected for RNA preparation. The FFPE blocks were cored with a sterile 16-gauge needle, and tumor areas showing at least 50% neoplastic cellularity were selected microscopically to enrich for neoplastic cells. Total RNA was extracted using the RecoverAll Total Nucleic Acid Isolation kit (Ambion Inc., Austin, TX, USA) per manufacturer’s protocol. RNA quality was evaluated using Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). All analyzed samples showed 28S to 18S ratio > 1.2, an RNA Integrity Number (RIN) > 8, and detectable microRNAs.
|
| Label |
Cy3
|
| Label protocol |
The miRNA Microarray System with miRNA Complete Labeling and hybridization Kit (Agilent Technologies) was used according to manufacturer′s protocols. The Agilent microRNA Spike-In Kit was used to measure labeling and hybridization efficiency. Briefly, 150ng total RNA was dephosphorylated at 37 degrees Celsius for 30 min with calf intestinal phosphatase and denatured by using 100% DMSO at 100 degrees Celsius for 7 minutes. Samples were labeled with pCp-Cy3 using T4 ligase by incubation at 16 degrees Celsius for 2 hour.
|
| |
|
| Hybridization protocol |
Hybridization were performed according to the Agilent miRNA Microarray Protocol (Agilent Technologies, Santa Clara, CA). Briefly, the labeled RNA samples were concentrated in a Vacuum Concentrator and prepared for hybridization by adding nuclease-free water, Hyb Spike-In solution, 10X GE Blocking Agent and 2X Hi-RPM Hybridization Buffer. 45 microliter of hybridization solution was dispensed into the gasket slide and assembled to the Agilent SurePrint G3 Human miRNA 8X60K, V16.0 Microarrays and then rotated at 20 rpm for 20 hours at 55 degrees Celsius.
|
| Scan protocol |
After hybridization, all slides were washed using Agilent Gene Expression Wash Buffers 1(at room temperature) and 2 ( at 37 degrees Celsius). The slides were immediately scanned on a Agilent Technologies G4900DA SureScan scanner at 3 micrometer resolution.
|
| Description |
Hybridization for patient S_12-36332 - Tumor
|
| Data processing |
Data were acquired with Agilent Feature Extraction 9.5.3.1 software for miRNA microarray, generating both probe-level signal intensities from all probes and summarized expression levels for each miRNA. The AgiMicroRna Bioconductor package was used to pre-process the raw data. First control, undetected probes, and outliers were filtered, then gene level expression summaries were obtained after normalization at the probe level using the RMA algorithm and quantile-normalization across samples.
|
| |
|
| Submission date |
Feb 26, 2017 |
| Last update date |
Feb 28, 2017 |
| Contact name |
Luigi Marchionni |
| E-mail(s) |
marchion@jhu.edu
|
| Phone |
410-502-8179
|
| Organization name |
Johns Hopkins University
|
| Department |
Oncology
|
| Lab |
Cancer Biology Program
|
| Street address |
1550 Orleans St., CRB2, Room 1M52
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21231 |
| Country |
USA |
| |
|
| Platform ID |
GPL16770 |
| Series (1) |
| GSE95385 |
Analysis of microRNA expression profiles in clear cell papillary renal cell carcinoma compared to normal adjacent tissue. |
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