|
| Status |
Public on Aug 01, 2011 |
| Title |
DAY 30 (sham) |
| Sample type |
RNA |
| |
|
| Source name |
Mus musculus kidney sham
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57/BL6
tissue: kidney
age: 10-12 weeks
gender: Female
stress: sham operated control
time: day 30
|
| Treatment protocol |
Mice were either 1)untreated naïve, 2) sham operated controls or 3) 30 minutes warm ischemia. Tissue was harvested from groups 2 and 3 on days 1,3,5,7,10,14,21 and 30
|
| Growth protocol |
Kidney tissue was extracted directly from treated mice
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from kidney tissue using the miRNeasy kit
|
| Label |
Cy5
|
| Label protocol |
The assay started from 4 to 8 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore) and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining; two different tags were used for the two RNA samples in dual-sample experiments
|
| |
|
| Hybridization protocol |
Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://microrna.sanger.ac.uk/sequences/) or other RNA (control or customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 μL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C. After RNA hybridization, tag-conjugating Cy3 and Cy5 dyes were circulated through the microfluidic chip for dye staining.
|
| Scan protocol |
Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
|
| Description |
Sample 8_Cy5
|
| Data processing |
Data were analyzed by LC sciences by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression)
|
| |
|
| Submission date |
May 24, 2011 |
| Last update date |
Aug 01, 2011 |
| Contact name |
John Iacomini |
| E-mail(s) |
jbagley1@tuftsmedicalcenter.org
|
| Organization name |
Tufts University/ Medical Center
|
| Street address |
800 Washington st
|
| City |
Boston |
| ZIP/Postal code |
02111 |
| Country |
USA |
| |
|
| Platform ID |
GPL13642 |
| Series (1) |
| GSE29495 |
Murine Kidney cells: Control versus Ischemia reperfusion injury |
|
