|
| Status |
Public on Sep 15, 2015 |
| Title |
APA of ADR032 |
| Sample type |
RNA |
| |
|
| Source name |
Conn's Syndrome_APA
|
| Organism |
Homo sapiens |
| Characteristics |
disease: Conn's Syndrome
patient/tissue id: ADR032
tissue: Human adrenal tissue
tissue subtype: Aldosterone-Producing Adenoma
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Adrenal collection protocol: The aldosterone-producing adenoma (APA) and their paired adjacent adrenal gland (for ZG and ZF) or adrenal gland cortex adjacent to pheochromocytomas were sanp-frozen by liquid nitrogen and collected from the Human Research Tissue Bank in Addenbrooke's hospital. Local ethical approval and informed consent were obtained for each patient.
Laser capture microdissection was used to acquire samples. For differentiation of ZG from ZF, sections were stained with cresyl violet using the LCM Staining Kit (AM1935, Ambion, USA). The acquired cell samples were stored in RNAlater (Ambion, USA) and TRIzol® reagent (Life Technologies, USA) at -70°C till extracted for RNA. Total DNA-free RNA was isolated using the PureLink® RNA Mini Kit and DNase Set (Life Technologies, USA) according to manufacturer’s instruction. Reverse transcription was performed using the Reverse Transcription System (Promega, USA) with a 1:1 mixture of random hexamer and oligo-dT primers according to manufacturer’s instruction. One RNA sample from the ZG of ADR121 was not analyzed as it did not pass quality control.
|
| Label |
biotin
|
| Label protocol |
Total RNA obtained through LCM were sent to Genomics Corelab, Cambridge Biomedical Research Centre for microarray measurement. Each RNA sample (total RNA, 1 µg) was converted to cDNA by reverse transcription. NuGEN Technologies’ Ovation Pico WTA System V2 kit was used to generate the first strand cDNA from total RNA. A DNA/RNA hetero-duplex-tagged double strand cDNA was then generated and amplified using a DNA/RNA chimeric primer (SPIA® primer), DNA polymerase and RNase H. This cDNA was fragmented by a chemical and enzymatic process using the NuGEN Technologies Encore Biotin Module to fragments between 50 and 100 base pairs in length. These fragmented products were then labeled via enzymatic attachment of a biotin-labeled nucleotide to the 3-hydroxyl ends of the fragments.
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| |
|
| Hybridization protocol |
Hybridization cocktails containing the fragmented and labeled cDNA and additional Affymetrix hybridization controls, were incubated on the Affymetrix arrays for 17hrs +/- 1hr at 45°C.
|
| Scan protocol |
GeneChips were scanned using the GeneChip® (GCS3000) Scanner.
|
| Description |
Gene expression data from snap-frozen human adrenals using liquid nitrogen and the cells for RNA extraction were collected by laser capture microdissection
Affy0257-37
|
| Data processing |
Data processing and analysis was performed using AffymetrixGeneChip Command Console Software and PartekGenomicSuite 6.5 (Partek Inc., St. Louis, MO).
Gene expressions were portrayed as the summarized log-signal of the Robust Multichip Average (RMA) with quantile normalisation and median polish for probe set summarisation.
|
| |
|
| Submission date |
Jan 14, 2015 |
| Last update date |
Sep 15, 2015 |
| Contact name |
Junhua Zhou |
| Organization name |
University of Cambridge
|
| Street address |
CPU, Level 6, ACCI, Addenbrooke’s Hospital
|
| City |
Cambridge |
| State/province |
Cambridgeshire |
| ZIP/Postal code |
CB2 0QQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10739 |
| Series (1) |
| GSE64957 |
Microarray study of human adrenal zona glomerulosa (ZG), zona fasciculata (ZF) and aldosterone-producing adenomas (APA) |
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