|
| Status |
Public on Mar 22, 2016 |
| Title |
3T3 0h - rep3 |
| Sample type |
RNA |
| |
|
| Source name |
NIH3T3 cells without treatment
|
| Organism |
Mus musculus |
| Characteristics |
cell line: NIH3T3
cell type: fibroblast
|
| Treatment protocol |
Asynchronously proliferating cells were challenged 2ug/ml tunicamycin for 2, 5 or 10 hrs.
|
| Growth protocol |
Cells were grown in DMEM containing 10% fetal bovine serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified using miRNeasy Mini Kit (QIAGEN)
|
| Label |
FlashTag™ Biotin
|
| Label protocol |
microRNA was labeled by FlashTag™ Biotin HSR RNA labeling kit (Affymetrix,CA). PolyA tailling buffer, ATP, PAP enzyme were mixed and incubated with RNA at 37°C for 15 min for 3' tailling. Then add the biotin ligation mix, T4 DNA ligase into tailed RNA, incubate them at 25°C for 30 min. Then stop the reaction by adding HSR stop solutions (Affymetrix, CA).
|
| |
|
| Hybridization protocol |
Add hybridation master mix (GeneChip® Eukaryotic Hybridization kit) into Biotin labled RNA. Incubate at 99°C for 5 minutes, then 45°C for 5 minutes.inject it into an array. Place the array into hybridization oven trays.Load the trays into the hybridization oven.Incubate the arrays at 48°C and 60 rpm for 16 to 18 hours.
|
| Scan protocol |
Wash, stain, and scan were following the Genechip® Expression User Guide for Cartridge Arrays (PN 702731). Scanning arrays was following the instructions in Affymetrix® Command Console® User Guide (P/N 702569).
|
| Description |
cells without treatment
|
| Data processing |
Results were analyzed using GENESPRING software. probe intensities were imported, normalized and summarized to the miRNA level using the RMA algorithm. RMA includes a background-subtracted probe-level quantile normalization prior to the median polish summarization to the miRNA log2-transformed intensities.
ANOVA and T-tests were used to calculate fold change and p-values. 1-way anova was performed. Low p-values identified genes that were not equal across all of the time points. 3 pairwise comparisons were performed, time points 2, 5 and 10 hours each vs the 0 hr. These comparison each gave a p-value and a fold change. All p-values were then corrected for multiple testing. Spotfire (Somerville, MA) software was used to generate heat maps.
|
| |
|
| Submission date |
Mar 21, 2016 |
| Last update date |
Mar 22, 2016 |
| Contact name |
John Alan Diehl |
| E-mail(s) |
diehl@musc.edu
|
| Organization name |
Medical University of South Carolina
|
| Street address |
86 Jonathan Lucas Street
|
| City |
Charleston |
| State/province |
SC |
| ZIP/Postal code |
29425 |
| Country |
USA |
| |
|
| Platform ID |
GPL14613 |
| Series (1) |
| GSE79455 |
Genome-wide microRNA expression profiling in tunicamycin treated NIH3T3 cells |
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