|
| Status |
Public on Apr 30, 2018 |
| Title |
leiomyoma B, Clariom D |
| Sample type |
RNA |
| |
|
| Source name |
50 year old woman, morcellated specimen of uterine fibroids after LASH
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: uterus
histology: leiomyoma
gender: female
|
| Treatment protocol |
Laparoscopic supracervical hysterectomy (LASH) due to multiple uterine fibroids. Further tumors were removed by laparotomy. For histologic examination tissues were fixed in paraformaldehyde (4% in PBS), and processed for paraffin embedding using standard techniques.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Simultaneous extraction and purification of DNA and RNA from FFPE tissue samples using Covaris adaptive focused acoutics (AFATM) and truXTRACTM FFPE DNA and RNA kits: The emulsification of the paraffin was done applying 5 minutes focused acoustic waves of 75 watts to a 10 mm FFPE section using a Covaris M220 device (Woburn, Massachusetts, USA). A Proteinase K digestions was performed at 56° C for 15 minutes. The paraffin including the genomic DNA was sedimented at 16.000 g. For RNA isolation the supernatant was heated to 80° C to reverse the formaldehyde crosslink followed by a DNaseI digestion step and a spincolumn cleanup procedure. Accordingly the pellet was subjected to spincolumn cleanup of the genomic DNA after an additional Proteinase K digestion. Fluorometric quantification was performed with the QubitTM 2.0 fluorometer using the QubitTM dsDNA HS-‐ and RNA HS Assay Kit (Thermo Fisher Scientific).
|
| Label |
biotin
|
| Label protocol |
For OncoScan FFPE assay, labelling of 80 ng dsDNA was done following the manufacturer’s instructions (Affymetrix P/N 703175). For Clariom D assay, 45 ng whole RNA was used for first strand cDNA synthesis is introducing by N6-‐ and Oligo-‐dT priming a T7 promoter sequence 5extended with an universal PCR primer site. To perform a preamplification (Pre-‐IVT Amplification) the primer site at the 5`ends of the first cDNA strand is countered by a second strand synthesis random primed (N6) with an adaptor attaching the reverse primer site to each end of the second strand fragments produced by Klenow polymerase. These templates were amplified exponentially for 6 cycles followed by a linear amplification step using the T7 promoter in an over night reaction (IVT, 14 h). Using 20 mg of each aRNA sample after purification as template in a reverse 3 transcription reaction a strand-‐identical single strand DNA was produced by adding random primers and dNTP`s. To enable an endpoint fragmentation reaction a certain fraction of dTTP is replaced by dUTP. After complete RNA removal (RNaseH) the enzymatic fragmentation is performed by uracil deglycosidase for removing uracil in combination with apurinic apyrimidinic endonuclease 1, which is breaking the free endonucleolytic phosphodiester bonds. Desoxynucleotidyl--‐transferase is adding Biotin--‐ 11--‐dXTP to the 3ends.
|
| |
|
| Hybridization protocol |
Prior to hybridization samples were denatured at 95°C. The hybridization was carried out at 49°C (OncoScan) or 45°C (Clariom D) in the GeneChipR Hybridization Oven 645 (Affymetrix) over night. Staining and washing was done using a GeneChip Fluidics Station 450 (Affymetrix) following the manufacturer’s instructions.
|
| Scan protocol |
Arrays were scanned using the GeneChipR Scanner 3000.
|
| Description |
transcriptome array data of a morcellated leiomyoma specimen
7065-13B
|
| Data processing |
Clariom D probe level analysis with SST/RMA (signal space transformation/robust multichip average) and data visualization was done with TAC (Transcriptome Analysis Console 4.0; Applied Biosystems).
|
| |
|
| Submission date |
Aug 24, 2017 |
| Last update date |
Apr 30, 2018 |
| Contact name |
Carsten Holzmann |
| E-mail(s) |
carsten.holzmann@med.uni-rostock.de
|
| Phone |
+493814947088
|
| Organization name |
University Rostock Medical Center
|
| Department |
Institute of Medical Genetics
|
| Street address |
Ernst-Heydemann-Str. 8
|
| City |
Rostock |
| ZIP/Postal code |
18055 |
| Country |
Germany |
| |
|
| Platform ID |
GPL23126 |
| Series (1) |
| GSE103050 |
Examination of tumor samples obtained after laparoscopic supracervical hysterectomy and a derivative leiomyosarcoma |
|
