|
| Status |
Public on Aug 04, 2016 |
| Title |
Nucleus pulposus_degenerated_3_circRNA |
| Sample type |
RNA |
| |
|
| Source name |
Nucleus pulposus tissue,degenerated
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Nucleus pulposus
condition: Degenerated
idd related diseases: Intervertebral disc herniation
gender: Male
age: 42 years
degeneration grading: Grading IV
disc level: L3-L4
|
| Treatment protocol |
All the specimens were collected from intervertebral discs of cadaveric donors or patients within 3 hours after anatomy or surgery respectively, following rinsed with phosphate-buffered saline, the nucleus pulposus tissue was carefully collected under stereoscopic microscope.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol (Invitrogen) and miRNeasy mini kit (QIAGEN) according to manufacturer’s instructions, which efficiently recovered all RNA species, including circRNAs. RNA quality and quantity was measured by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies) and RNA Integrity was determined by gel electrophoresis
|
| Label |
Cy3
|
| Label protocol |
CcircRNA were treated with Rnase R (Epicentre, Inc.) to remove linear RNAs. Then, each sample was amplified and transcribed into fluorescent cRNA utilizing a random priming method (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000.
|
| |
|
| Hybridization protocol |
1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60 °C for 30 min, finally 25 μl 2 × Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the circRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.
|
| Scan protocol |
The hybridized arrays were washed, fixed and scanned using the Agilent Scanner G2505C.
|
| Description |
Same RNA as GSM1354771, GSM1551031
|
| Data processing |
Scanned images were imported into Agilent Feature Extraction software for raw data extraction. Quantile normalization of raw data and subsequent data processing were performed using the R software package. After quantile normalization of the raw data, low intensity filtering was performed, and the circRNAs that at least 5 out of 10 samples have flags in “P” or “M” (“All Targets Value”) were retained for further analyses.
When comparing two groups of profile differences (such as disease versus control), the “fold change” (i.e. the ratio of the group averages) between the groups for each circRNA is computed. The statistical significance of the difference may be conveniently estimated by t-test. circRNAs having fold changes >2 and p-values < 0.05 are selected as the significantly differentially expressed.
|
| |
|
| Submission date |
Apr 03, 2015 |
| Last update date |
Aug 04, 2016 |
| Contact name |
Hai-Qiang Wang |
| E-mail(s) |
hqwang@sntcm.edu.cn, drwanghq@163.com
|
| Organization name |
Shaanxi University of Chinese Medicine
|
| Department |
Insititute of Integrative Medicine
|
| Street address |
Xixian Avenue, Xixian District
|
| City |
Xi'an |
| ZIP/Postal code |
712046 |
| Country |
China |
| |
|
| Platform ID |
GPL19978 |
| Series (2) |
| GSE67566 |
The expression profiling of circular RNAs (circRNA) in human intervertebral disc degeneration (IDD) |
| GSE67567 |
Noncoding RNAs in human intervertebral disc degeneration: an integrated microarray study |
|
| Relations |
| Alternative to |
GSM1354771 (identical RNA) |
| Alternative to |
GSM1551031 (identical RNA) |
