|
| Status |
Public on Apr 01, 2022 |
| Title |
344-TS: Tumour stroma_sample_344 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Invasive Lobular Carcinoma fresh frozen tumor
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Tumour stroma
patient age: 75
tumor size: 21
nuclear grade: II
er status: pos
pr status: pos
her2 status: neg
|
| Treatment protocol |
Tissue specimens were microdissected into epithelium and stroma using a PixCell IIe LCM system (Arcturus). All microdissections were performed within two hours following tissue staining.
|
| Growth protocol |
Breast tumor tissue samples were obtained at primary surgery from patients providing written informed consent.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each population of microdissected cells in RNase-free eppendorf containing 50 µl of extraction buffer and placed for 30 min at 42 ºC (Arcturus). After RNA extraction, RNA was isolated and subjected to DNaseI treatment (PicoPure RNA isolation kit, Arcturus). Samples were concentrated to a final volume of 6 µl using a refrigerated CentriVap concentrator (LabConco) at 4 ºC for 10 min. Agilent RNA 6000 Pico kit was used for Bioanalyzer quality control (Agilent).
|
| Label |
Cy3
|
| Label protocol |
RNA was amplified using the T-7 based RiboAmp HS Plus RNA amplification kit (Arcturus) and quality control Agilent RNA 6000 Nano kit was used (Agilent). RNA was labelled with Cy3 dye (Turbo labeling kit Cy3, Life Technologies) according to the manufacturer’s procedure. The amount of Cy3-labeled aRNA was quantified using a NanoDrop.
|
| |
|
| Channel 2 |
| Source name |
Universal Human Reference RNA Sample
|
| Organism |
Homo sapiens |
| Characteristics |
supplier: Stratagene, #740000, La Jolla, California, USA
|
| Treatment protocol |
Tissue specimens were microdissected into epithelium and stroma using a PixCell IIe LCM system (Arcturus). All microdissections were performed within two hours following tissue staining.
|
| Growth protocol |
Breast tumor tissue samples were obtained at primary surgery from patients providing written informed consent.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from each population of microdissected cells in RNase-free eppendorf containing 50 µl of extraction buffer and placed for 30 min at 42 ºC (Arcturus). After RNA extraction, RNA was isolated and subjected to DNaseI treatment (PicoPure RNA isolation kit, Arcturus). Samples were concentrated to a final volume of 6 µl using a refrigerated CentriVap concentrator (LabConco) at 4 ºC for 10 min. Agilent RNA 6000 Pico kit was used for Bioanalyzer quality control (Agilent).
|
| Label |
Cy5
|
| Label protocol |
RNA was amplified using the T-7 based RiboAmp HS Plus RNA amplification kit (Arcturus) and quality control Agilent RNA 6000 Nano kit was used (Agilent). RNA was labelled with Cy3 dye (Turbo labeling kit Cy3, Life Technologies) according to the manufacturer’s procedure. The amount of Cy3-labeled aRNA was quantified using a NanoDrop.
|
| |
|
| |
| Hybridization protocol |
SurePrint G3 Human GE 8x60K microarray kit with SurePrint technology (Agilent Technologies, #G4851-60510) were used for all the experiments. Two-color microarray-based gene expression hybridization kit was used to perform the hybridizations (Agilent, #5188-5242). The 2 dyes used were Cy3 and Cy5. The aRNA of interest (300 ng) was labelled with Cy3. The reference sample used (300 ng) was a universal human reference RNA sample (Stratagene, #740000, La Jolla, California, USA) that was previously amplified using the RiboAmp HS plus kit and labelled with Cy5 dye using the Arcturus Cy5 Turbo Labeling Kit (Life Technologies, #KIT0610).
|
| Scan protocol |
Microarray data were feature extracted using Feature Extraction Software (v. 9.5.3) from Agilent with the default parameters.
|
| Data processing |
The microarray experiments were carried out in 3 batches and pre-processed using Agilent GeneSpring Software. Raw data was normalised, log2 transformed and filtered, and quality control analyses were carried out. HGNC gene annotations were applied and the mean was taken when multiple probes were present for the same gene. Finally, the batch effect was corrected using ComBat.
|
| |
|
| Submission date |
Apr 09, 2020 |
| Last update date |
Apr 01, 2022 |
| Contact name |
Andrew H Sims |
| E-mail(s) |
andrew.sims@ed.ac.uk
|
| Organization name |
University of Edinburgh
|
| Department |
Institute of Genetics and Molecular Medicine
|
| Lab |
Applied Bioinformatics of Cancer
|
| Street address |
Carrington Crescent
|
| City |
Edinburgh |
| ZIP/Postal code |
EH4 2XR |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL14550 |
| Series (1) |
| GSE148398 |
Gene expression of invasive lobular carcinoma tumor epithelium and tumor stroma isolated by laser-capture microdissection |
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