|
| Status |
Public on Jan 01, 2023 |
| Title |
SCI, 3 days, non-SPF, replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Spinal cord tissue, treated with a modified Allen’s weight-drop apparatus
|
| Organism |
Rattus |
| Characteristics |
strain: Sprague-Dawley
tissue: Spinal cord tissue
Sex: male
treatment: Spinal cord injury
|
| Treatment protocol |
Three rat SCI models was established by the modified Allen method. Rats were anesthetized by inhalation of isoflurane and were placed in the prone position on an operating table. The head and limbs were held in place and the fur along the back was removed for skin preparation. After strict disinfection, a median incision of approximately 3 cm in length was made at the T10 level. The skin, subcutaneous tissue, and myofascia were carefully separated and dissected, exposing and fenestrating the T10 lamina. The spinal cord was carefully exposed without damaging the dura mater. A heavy hammer weighing 10 g with a diameter of 2.5 mm was allowed to fall freely from a height of 8 cm, fully impacting the dural sac; the hammer was then retracted immediately after SCI. After the blow, the rats show a tail-wagging reflex with twitching and stretching of both hind limbs and the body; this is a stress reflex and indicates the success of the modeling. The sham operation group received the same procedure without injury to the spinal cord.
|
| Growth protocol |
Studies were conducted using adult Sprague-Dawley rats (male, 200-250g) that had been housed under standard conditions (23-25°C, 12 h light/dark cycle) for at least one week before experimental use with free access to food and water.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted by mirVanaTM RNA Isolation Kit (Applied Biosystem p/n AM1561 ) following the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 μg RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
0.6 μg of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/μg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 22.5μl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers' instructions. On completion of the fragmentation reaction, 22.5μl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Human Gene Expression（8*60K）for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4x180k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after treated with a modified Allen’s weight-drop apparatus spinal cord tissue
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities as the raw data. Raw data were normalized in quantile algorithm with Genespring 13.0(Agilent). Probe that at least 1 out of 2 samples flagged as Detected were maintained.
|
| |
|
| Submission date |
Nov 16, 2022 |
| Last update date |
Jan 02, 2023 |
| Contact name |
Jian Cao |
| E-mail(s) |
361439920115@email.ncu.edu.cn
|
| Organization name |
The Second Affiliated Hospital of Nanchang University
|
| Department |
orthopaedics
|
| Street address |
1 Minde Road, East Laker District, Nanchang, Jiangxi, China
|
| City |
Nanchang |
| State/province |
Jiangxi |
| ZIP/Postal code |
330000 |
| Country |
China |
| |
|
| Platform ID |
GPL30293 |
| Series (1) |
| GSE218088 |
Expression Profiles of Long Non-Coding RNAs and Messenger RNAs in a Rat Model of Spinal Cord Injury |
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