|
| Status |
Public on Jan 01, 2008 |
| Title |
Periprosthetic_membrane_infectious_CodeAB88 |
| Sample type |
RNA |
| |
|
| Source name |
Periprosthetic membrane, infectious type, internal code number AB88
|
| Organism |
Homo sapiens |
| Characteristics |
Gender: Male
Age: 81 years
Time between primary arthroplasty and revision surgery: 5.1 years
Microbiological culturing result: Streptococcus orales
Localization: Right knee joint
Cementation: Yes
|
| Biomaterial provider |
Center for musculoskeletal surgery, Charite University Hospital Berlin
|
| Treatment protocol |
The samples was excised during endoprosthetic revision surgery. Inside the operation room, the sample was macroscopically described, measured, and cut into pieces of 3x3x3 mm. One piece was fixed in 4% buffered formalin, and the immediately adjacent piece was snap frozen in liquid nitrogen and then stored at -80 degrees Celsius. HE stained section was made from the formalin fixed tissue. As the HE slide showed the typical morphology of an endoprosthesis loosening membrane, the adjacent kryopreserved sample was used for RNA-extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol (INVITROGEN) followed column based clean up (RNeasy, QIAGEN). Total RNA was quantified by UV-spectroscopy and its quality was checked by analysis on a LabChip (BioAnalyzer, AGILENT TECHNOLOGIES). Three micrograms from each sample were synthesized into double-stranded cDNA using SuperScript transcriptase (LIFE TECHNOLOGIES, INC.) and with an oligo(dT)24 primer containing a T7 RNA polymerase promoter (TIBMOL BIOL). After second strand cDNA synthesis (LIFE TECHNOLOGIES, INC.), the product was purified and served as template in the subsequent in vitro transcription reaction.
|
| Label |
Biotin
|
| Label protocol |
Labeled complementary RNA (cRNA) was prepared from double-stranded cDNA by in vitro transcription using the GeneChip RNA transcript labeling kit (AFFYMETRIX) according to the manufacturer’s instruction. After cleanup (QIAGEN), the biotin-labeled cRNA was fragmented by alkaline treatment (40 mmol/L Tris-acetate (pH 8.2), 100 mmol//L potassium acetate, and 50 mmol//L magnesium acetate) at 94 degrees Celsius for 35 minutes.
|
| |
|
| Hybridization protocol |
15 micrograms of each fragmented cRNA sample was hybridized for 16 hours at 45 degrees Celsius to an Affymetrix HG-U133A GeneChip Array. Chips were washed and stained with streptavidin-phycoerythrin using a fluidics station according to protocols recommended by Affymetrix.
|
| Scan protocol |
Probe arrays were scanned at 3 micrometers resolution using the Affymetrix GeneChip System confocal scanner 2500 with argon laser instrument, controlled by Microarray Suite 5.0 software (AFFYMETRIX). After a scan of the array surface, the computer-generated image of the array was overlayed with a virtual grid, controlled by Microarray Suite 5.0 software. This allowed for each feature to be defined, and the interrogating features aligned to known dimensions of the array.
|
| Description |
The features at the corner- and edge-regions were control markers. About 80 pixels within each feature were averaged after discarding outliers and pixels near feature boundaries. The quantitative expression level for each gene was calculated by the average differences representing the perfectly matched versus the mismatched specific probe. Arrays were scaled to an average intensity of 500 degrees per gene and analyzed independently. Text files were exported for the intensity information for each interrogating oligonucleotide.
|
| Data processing |
The Data Mining Tool 3.0 (AFFYMETRIX) and GeneSpring software package 6.1 (SILICON GENETICS) were used to average results from different replicates and perform statistical analysis to compare between treatments. The data were normalized to account for variability in hybridization for probe pairs and other hybridization artifacts. The normalization consists of the following 3 steps: first, data transformation (set measurements less than 300.0 to 300.0), second, per chip (normalize each chip to the 50th percentile of the measurements taken from that chip) and third, per gene (normalize each gene to the median of the measurements for that gene).
|
| |
|
| Submission date |
Feb 22, 2007 |
| Last update date |
Feb 23, 2007 |
| Contact name |
Lars Morawietz |
| E-mail(s) |
[email protected]
|
| Phone |
+4930450536174
|
| Fax |
+4930450536907
|
| Organization name |
Charite University Hospital
|
| Department |
Institute for Pathology
|
| Street address |
Chariteplatz 1
|
| City |
Berlin |
| ZIP/Postal code |
D-10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE7103 |
Gene expression profiling in wear-particle induced and infectious endoprosthesis loosening |
|
