|
| Status |
Public on Nov 26, 2019 |
| Title |
Rectal biopsy 30 |
| Sample type |
RNA |
| |
|
| Source name |
Frozen rectal cancer biopsy
|
| Organism |
Homo sapiens |
| Characteristics |
subject status: patient with rectal adenocarcinoma
ajcc score: 0
gender: Male
overall survival (in days): 2960
dead (1)/alive(0): 0
age: 62
|
| Treatment protocol |
Freshly harvested, the biopsies were snap frozen at -80°C. The frozen biopsies were then embadded in medium for frozen specimen to ensure optimal cutting temperature (O.C.T). Per biopsy, 12 slices of 10µm each were cut and used for mRNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The mRNA were extracted from the 12 slices/biopsy using the RNAqueous Kit (Ambion, Austin, Texas, USA) according the manufacturer's protocol. Then samples were treated with TURBO DNA‐free (Ambion, Austin, Texas, USA) to ensure a complete degradation of genomic DNA. Concentrations of mRNAs were determined with Nanodrop (ThermoFisher, Waltham, Massachusetts, USA) and quality of the extractions was assessed by 260/280nm readings. Integrity of mRNAs was verified by migration on agarose gel 1%.
|
| Label |
Cy3
|
| Label protocol |
The Biotin-labeling was done during cRNA convertion using the TotalPrepTM RNA amplification kit (Illumina, San Diego, California, USA) . Briefly, 100ng of mRNAs were used for a two-steps reverse transcription reaction and being converted into cDNA. After elution, the cDNA are converted into cRNA using biotin-NTP mix and then purified with a filter cartbridge. After hybridization and blockage, Biotin-labeled cRNA are incubated in streptavidin-Cy3 buffer provided in the kit at room temperature for 10 min and then washed with E1BC buffer provided in the kit and dried.
|
| |
|
| Hybridization protocol |
1.5µg of biotinylated cRNA were incubated 5 minutes at 65°C before being mixed with 20µL hybridation mix provided by the kit (Illumina, San Diego, California, USA). After being placed into the chamber, the beadchip were loaded with 30uL of cRNA per sample. The chamber is placed 17hrs at 58°C. Beadchips are then washed with 250mL of the E1BC solution (Illumina) before a second wash at high temperature (55°C). The beadchips are rinced with fresh E1BC buffer then submerged for 10min with 100% ethanol. The beadcips are washed for a second time at room temperature and blocked with buffer provided by the kit.
|
| Scan protocol |
Slides were scanned immediately after washing on the Beadarray Reader (illumina) with Sentrix type 6x2and Direct hyb settings. Dye chennal is set to green with green PMT sets to 100% and gain to 0.5. After measurment of the tilt and alignment of the beadchips, scann is completed and .Tiff uncompressed images are then generated
|
| Description |
Gene expression in pretreated rectal cancer biopsy measured in frozen tissue
|
| Data processing |
The scanned images with .tiff format were analyzed with Beadstudio Gene expression module v3.4 using default parameters tu quantify floresence intensity and substract the background for each beadchips analyzed.
|
| |
|
| Submission date |
Jun 20, 2019 |
| Last update date |
Nov 26, 2019 |
| Contact name |
Matthew Kalady |
| Phone |
(+1)216-445-9755
|
| Organization name |
CLEVELAND CLINIC
|
| Department |
Cancer Biology Department
|
| Street address |
9500 EUCLID AVE
|
| City |
CLEVELAND |
| State/province |
OHIO |
| ZIP/Postal code |
44195 |
| Country |
USA |
| |
|
| Platform ID |
GPL6102 |
| Series (1) |
| GSE133057 |
Transcriptomic analysis of pretreated rectal cancer biopsies and association to the tumor regression score. |
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