tissue: LIDH patients(EG) Sex: Male age: 63 years segment: L4/5 mri grade: V
Extracted molecule
total RNA
Extraction protocol
Total RNA from each sample was isolated individually using the TRIzol (Invitrogen, Carlsbad, CA, USA) and miRNeasy mini kit (QLAGEN, Valencia, CA, USA) according to manufacture's instructions, which efficiently recovered all RNA species, including miRNAs. RNA quality and quantity was measured by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies, Wilmington, DE, USA) and RNA integrity was determined by gel electrophoresis.
Label
Hy3
Label protocol
After RNA isolation from the samples, the miRCURY Hy3/Hy5 Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer's guideline for miRNA labelling. One microgram of each sample was 3'-end-labeled with Hy3TM fluorescent label, using T4 RNA ligase. After stopping the labeling procedure, the Hy3TM-labeled samples were hybridized on the miRCURYTM LNA Array (v.16.0) (Exiqon) according to array manual.
Hybridization protocol
The total mixture with hybridization buffer was hybridized to the microarray in a 12-Bay Hybridization System (Hybridization System, NimbleGen Systems, Inc., Madison, WI, USA), which provides an active mixing action and a constant incubation temperature to improve hybridization uniformity and enhance signal.
Scan protocol
Following hybridization, the slides were washed several times using the wash buffer kit (Exiqon), and finally dried by centrifugation. The slides were then scanned with the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA, USA).
Data processing
For each spot, verify the signal by subtracting background intensity (B) from foreground intensity (F). Verified signal value= F - B. The spots, of which verified signal values are less than 50, and the control spots are filtered. For verified signal values of the left spots, perform median normalization.
MicroRNA Expression Profiles identify genes of apoptosis, anabolism and catabolism in patients with Intervertebral Disc Degeneration different from Spinal Trauma
