|
| Status |
Public on May 26, 2011 |
| Title |
astrocytoma_RISC-IP microRNA (02_H11.0_092343) |
| Sample type |
RNA |
| |
|
| Source name |
human U-87 MG astrocytoma/glioblastoma cell line (ATCC, cat# HTB-14)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: human U-87 MG astrocytoma/glioblastoma cell line (ATCC, cat# HTB-14)
passage number: between 18 - 22
cell fraction: RISC-immunoprecipitated
antibody: anti-RISC
|
| Growth protocol |
Primary human cerebral cortex astrocyte cells were cultured in cell specific astrocyte medium containing 2% fetal bovine serum, 1% astrocyte growth supplement and 1% penicillin/streptomycin (ScienCell Research Laboratories). Adherent human U-87 MG astrocytoma/glioblastoma cells were cultured under 5% CO2 in DMEM F12 + 1% L-glutamine (2 mM) (Cambrex, Walkersville, MD) supplemented with 10% fetal calf serum (Gibco, Burlington, ON, Canada), 1% penicillin-streptomycin (Gibco), 1% sodium pyruvate (1 mM) (Gibco) and 1% non-essential amino acids (0.1 mM) (Gibco) at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA enriched with small RNAs was isolated from the RISC-IP complex and the global cellular fraction using the mirVana kit (Ambion) according to the procedures outlined in the manufacturer’s protocol.
|
| Label |
Cy5
|
| Label protocol |
The microarray assay was performed using a 5 µg total RNA sample, which was fractionated using a YM-100 Microcon centrifugal filter (Millipore, Billerica, MA) to isolate small RNAs (< 300 nt). Small RNAs 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 with Cy5.
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| |
|
| Hybridization protocol |
Hybridizations were performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies, Houston, TX). Each detection probe on the microfluidic chip consisted of a chemically modified nucleotide coding segment complementary to 6211 target human miRNAs (Sanger miRBase version 11, http://microrna.sanger.ac.uk/sequences/) or control RNAs (array hybridization controls and single-base mismatch targets), and a polyethylene glycol spacer segment to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using photogenerated reagent chemistry and were replicated seven times within each microarray chip [46]. The hybridization melting temperatures of the probes were balanced by chemical modifications of the detection probes. Hybridization was performed at 34 °C using 100 µL of 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA pH 6.8) containing 25% formamide.
|
| Scan protocol |
After washing, the hybridized fluorescent Cy5 signals were detected with a laser scanner (GenePix 4000B, Molecular Devices, Sunnyvale, CA) and digitized by Array-Pro image analysis software (Media Cybernetics, Bethesda, MA).
|
| Description |
The concentration of RNA samples was determined using Nanodrop ND-1000 Spectrophotometer and the RNA quality of 2 samples was checked on RNA Nano chip using Agilent 2100 Bioanalyzer. All RNA samples displayed no degradation with two sharp ribosomal peaks. The microarray assay was performed using a 5 µg total RNA sample, which was fractionated using a YM-100 Microcon centrifugal filter (Millipore, Billerica, MA) to isolate small RNAs (< 300 nt). Small RNAs 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 with Cy5.
|
| Data processing |
Data were further processed by subtracting the background followed by signal normalization with a LOWESS (locally weighted regression) filter.
For a miRNA transcript to be classified as reliably detectable, it had to satisfy the following criteria: overall signal intensity >3× background standard deviation, spot coefficient of variation <0.5, and >50% of the repeated probes had signal intensities above detection level.
|
| |
|
| Submission date |
Apr 26, 2010 |
| Last update date |
May 26, 2011 |
| Contact name |
Joanna Moser |
| Organization name |
University of Calgary
|
| Department |
Biochemistry and Molecular Biology
|
| Lab |
Dr. Marvin Fritzler
|
| Street address |
3330 Hospital Drive N.W.
|
| City |
Calgary |
| State/province |
AB |
| ZIP/Postal code |
T2N 4N1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL10358 |
| Series (1) |
| GSE21514 |
The mRNA and microRNA expression profile of the RNA-induced silencing complex in human U-87 astrocytoma cells and primary human astrocytes |
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