|
| Status |
Public on Dec 19, 2013 |
| Title |
sample collected from patient 1B ≤2 months after transplantation |
| Sample type |
RNA |
| |
|
| Source name |
deteriorated renal graft function_≤2 months
|
| Organism |
Homo sapiens |
| Characteristics |
patient id: 1B
time after transplantation: ≤2 months
deterioration of graft function within 2 years after transplantation: yes
sample type: renal graft biopsy
|
| Treatment protocol |
Comparison of patients with stable and deteriorated renal graft function (defined as as the increase in serum creatinine level of more than 25% of its value at 3 months) during 2 years afte transplantation by analyzing RNA profiles at the time of border line changes diagnosis in early (≤2 months) or protocolar (3 months) renal graft biopsies.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The small portion (2mm) of the the renal graft biopsy was immediately placed in RNA later (Ambion Corporation, Austin, TX), snap frozen and stored at -80 °C until RNA extraction. RNA was isolated from renal biopsies using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany).Quality and integrity of PAXgene RNA were determined using the Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was quantified by measuring absorbance at 260 nm on the ND-1000 Spectrophotometer (NanoDrop Technologies).
|
| Label |
Cy3
|
| Label protocol |
RNA was amplified and biotinylated for hybridization using Ambion® Illumina® TotalPrep™ RNA Amplification Kit according to manufacturer´s instructions. For all samples 200 ng of total RNA was used for amplification. Yields of labeled cRNA were determined using the Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| |
|
| Hybridization protocol |
For all samples a total of 750 ng of labeled cRNA was hybridized at 58°C for 16 hours to the Illumina HumanHT-12 v4.0 Expression BeadChips (Illumina) and subsequently washed.
|
| Scan protocol |
Fluorescence signals of the hybridized IlLumina Microarrays were detected using BeadStation 500 instrument (Illumina Inc). The raw data were extracted with the BeadStudio Data Analysis Software (Illumina).
|
| Description |
BORDERLINE_PRAGUE_1B
|
| Data processing |
Bead level data were summarized by Illumina BeadStudio Software v3, imported into R and normalized using the quantile method in the Lumi package. Please note that only the probes (23,719 ID_REFs) that were expressed in the biopsy renal allograft samples are chosen for data normalization.
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| |
|
| Submission date |
Nov 24, 2013 |
| Last update date |
Dec 19, 2013 |
| Contact name |
Petra Hruba |
| E-mail(s) |
hrup@medicon.cz
|
| Phone |
+420236052148
|
| Organization name |
Institute for Clinical and Experimental Medicine
|
| Department |
Transplant Laboratory
|
| Street address |
Videnska 1958
|
| City |
Praha 4 |
| ZIP/Postal code |
140 21 |
| Country |
Czech Republic |
| |
|
| Platform ID |
GPL10558 |
| Series (1) |
| GSE52694 |
Borderline Changes in Renal Allografts: Molecular Diagnostics Identifies Risks for Graft Dysfunction |
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