|
| Status |
Public on Feb 01, 2015 |
| Title |
Primary Melanoma_035 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Cutaneous primary melanoma
|
| Organism |
Homo sapiens |
| Characteristics |
identifier: 035
gender: F
age: 30
primary tumor thickness (mm): 8
mitotic index: Few
ulceration status: Absent
histotype: Desmoplastic
anatomical site of primary tumor: Axial
Stage: IIB
recurrence: Y
time to 1st recurrence (days): 1945
time to 1st recurrence (months): 63
site of 1st recurrence: DV
brain as isolated 1st site of recurrence: N
b-met: N
time to b-met (days): NA
follow-up time (days): 2546
follow-up time (months): 83
last status: Alive, No Melanoma
Cause of death: NA
|
| Treatment protocol |
After surgical excision, primary melanomas were fixed in formalin and embedded in parrafin by NYU Langone's clinical pathology lab. 5uM FFPE sections were affixed to Leica PEN membrane slides. 3 tp 10 sections were used for RNA extraction from each patient case. A consecutive section was stained by H&E and tumor area was marked by an attending pathologist. Tumor was macro-dissected from sections under a dissecting microscope and transferred to microfuge tubes prior to RNA extraction. RNA extraction was performed within 4 days of tissue section to reduce air-related RNA degradation
|
| Growth protocol |
N/A
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Qiagen miRNeasy FFPE kits following the manufacturer's recommended protocol. RNA quantity and quality was assessed by Nanodrop and Agilent 2100 Bioanalyzer.
|
| Label |
Cy3 (Hy3)
|
| Label protocol |
300 ng total RNA from sample and reference was labelled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA microRNA Hi-Power Labelling Kit, Hy3/Hy5 (Exiqon, Denmark) following the procedure described by the manufacturer.
|
| |
|
| Channel 2 |
| Source name |
Common Reference
|
| Organism |
Homo sapiens |
| Characteristics |
material: Equal mixture of all samples
|
| Treatment protocol |
After surgical excision, primary melanomas were fixed in formalin and embedded in parrafin by NYU Langone's clinical pathology lab. 5uM FFPE sections were affixed to Leica PEN membrane slides. 3 tp 10 sections were used for RNA extraction from each patient case. A consecutive section was stained by H&E and tumor area was marked by an attending pathologist. Tumor was macro-dissected from sections under a dissecting microscope and transferred to microfuge tubes prior to RNA extraction. RNA extraction was performed within 4 days of tissue section to reduce air-related RNA degradation
|
| Growth protocol |
N/A
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Qiagen miRNeasy FFPE kits following the manufacturer's recommended protocol. RNA quantity and quality was assessed by Nanodrop and Agilent 2100 Bioanalyzer.
|
| Label |
Cy5 (Hy5)
|
| Label protocol |
300 ng total RNA from sample and reference was labelled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY™ LNA microRNA Hi-Power Labelling Kit, Hy3/Hy5 (Exiqon, Denmark) following the procedure described by the manufacturer.
|
| |
|
| |
| Hybridization protocol |
The Hy3™-labeled samples and a Hy5™-labeled reference RNA sample were mixed pair-wise and hybridized to the miRCURY™ LNA microRNA array (6th gen - hsa, mmu & rno) (Exiqon, Denmark), which contains capture probes targeting all miRNAs for human, mouse or rat registered in the miRBASE version 16.0 at the Sanger Institute. The hybridization was performed according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridization station (Tecan, Austria). After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes.
|
| Scan protocol |
The miRCURY™ LNA array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene 9.0 software (BioDiscovery, Inc., USA).
|
| Description |
1_Exiqon is Cy3
|
| Data processing |
The quantified signals were background corrected (Normexp with offset value 10 – Ritchie et al., 2007) and normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. Spots were flagged as follows: 0 - no flag, spot OK; 1 - manual flagging; 2 - empty spot; 3 - poor spot; 4 - negative spot; 5 - empty spot (manual); poor spot (manual); negative spot (manual). Spots flagged 1 or higher were removed from consideration. Capture probes for which both Hy3 and Hy5 values were less than 1.5x the median signal intensity for a given slide were excluded from further analysis. Excluded capture probes are indicated byt the acronym' NA' in the expression matrix. For each spot, a log base 2 ratio (Hy3/Hy5) was calculated, and the median log-ratio of four replicate spots per array was selected as representative of relative miRNA expression for a given sample. Median log-ratios were used for all subsequent analyses.
|
| |
|
| Submission date |
Oct 15, 2014 |
| Last update date |
May 09, 2015 |
| Contact name |
Eva Hernando-Monge |
| E-mail(s) |
Eva.Hernando-Monge@nyumc.org
|
| Phone |
212-263-9054
|
| Organization name |
NYU Langone Medical Center
|
| Street address |
550 1st Ave. Smilow 305
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10016 |
| Country |
USA |
| |
|
| Platform ID |
GPL19296 |
| Series (2) |
| GSE62371 |
miRNA expression profiling of primary melanoma tumors (cohort II) |
| GSE62372 |
miRNA expression profiling of primary melanoma tumors |
|
