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| Status |
Public on Aug 21, 2017 |
| Title |
Tomatidine_1 |
| Sample type |
RNA |
| |
|
| Source name |
WT worm_tomatidine
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
age: D7
treatment: Tomatidine_25 micromolar
|
| Treatment protocol |
N2 worms from the L4 stage were exposed to either vehicle (DMSO) or 25 μM tomatidine, and were collected on adult Day 7 by washing off the plates with M9 buffer.
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| Growth protocol |
Caenorhabditis elegans (C. elegans) nematodes were cultured at 25°C on solid agar plates of standard nematode growth medium (NGM) with OP50 food source unless otherwise, and maintained following standard protocols previously described. The Bristol strain (wild type) and CB5600 ccIs4251[pSAK2 (myo-3p::gfp-lacz(NLS)) + psak4 (myo-3p::mitochondrial gfp)I were from Caenorhabditis Genetics Center (CGC). The mitophagy reporter strain N2;Ex(pmyo-3::dsred::lgg-1;pdct-1::dct-1::gfp) was from Prof. N. Tavernarakis (University of Crete, Greece).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Four independent experiments per condition were collected, and stored at -80oC in lysis reagent (Qiagen Cat. no. 75142). Crude extracts were prepared by bead beating packed worm pellets with the Mini-Beadbeater 8 (Biospec Products, Oklahoma), and total RNA isolated using RNeasy Mini Kit on the QIAcube according to the manufacturer's protocol (Qiagen, California). RNA concentration and quality was measured by Nanodrop (ThermoFisher, Waltham, MA USA) and the Agilent Bioanalyzer RNA 6000 Chip (Agilent, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng total RNA using the Low-Input Quick Amp Labeling Kit (Agilent, Santa Clara, CA) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were evaluated with the NanoDrop ND-1000 Spectrophotometer (Wilmington, DE).
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| |
|
| Hybridization protocol |
A total of 825 ng Cy3-labeled cRNA (specific activity >6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 microL containing 0.5x Agilent fragmentation buffer and 2x Agilent gene expression blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 microL of 2× Hi-RPM Hybridization Buffer was added to the fragmentation mixture and hybridized to Agilent C. elegans (V2) 4×44K Gene Expression Microarrays (G2519F-020186) 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), and dried by slowly removing from Wash buffer 2. A total of 1.65 micrograms Cy3-labelled cRNA
|
| Scan protocol |
Following posthybridization rinses, arrays were scanned using an Agilent SureScan microarray Scanner at 5 micron, and hybridization intensity data extracted from the scanned images using Agilent’s Feature Extraction Software 9.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| Description |
TOM 300
|
| Data processing |
Raw microarray hybridization intensity data from four separate experiments were log-transformed. Three separate probe preparations were used individually or combined in equal amounts for the fourth hybridization. Raw microarray data were log transformed to yield z-scores. The z-ratio was calculated as the difference between the observed gene z-scores for the experimental and the control comparisons, and dividing by the standard deviation associated with the distribution of these differences. Z-ratio values of ± 1.5 were used as cut-off values and calculated using a 5% false discovery rate (FDR) threshold. The complete set was tested for gene set enrichment using parametric analysis of gene set enrichment. For each pathway z-score, a p-value was computed using JMP 6.0 software to test for the statistical significance.
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| |
|
| Submission date |
Aug 05, 2016 |
| Last update date |
Jun 22, 2020 |
| Contact name |
Supriyo De |
| Organization name |
NIA-IRP, NIH
|
| Department |
Laboratory of Genetics and Genomics
|
| Lab |
Computational Biology & Genomics Core
|
| Street address |
251 Bayview Blvd
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21224 |
| Country |
USA |
| |
|
| Platform ID |
GPL10094 |
| Series (1) |
| GSE85237 |
Tomatidine enhances lifespan and healthspan in C. elegans through mitophagy induction via the SKN-1/Nrf2 pathway. |
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