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Status |
Public on Jun 01, 2017 |
Title |
DNAN-62.5ppm-1 |
Sample type |
RNA |
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Source name |
Wild type C. elegans var. Bristol strain N2
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Organism |
Caenorhabditis elegans |
Characteristics |
tissue: Pooled somatic tissue of approx. 400 nematodes gender: hermaphrodite developmental stage: Late L3 stage grown from synchronized culture 24-hr acute treatment: DNAN concentration: 62.5ppm replicate: 1
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Treatment protocol |
After harvesting from agar plates, worms were spun at 1150 x g for 2.5 min and re-suspended in a given volume of K Medium. Twenty µL of worms were deposited on a microscope slide containing a grid sticker and worms were counted. The number of worms/mL was then calculated. Wide-bore pipet tips or transfer pipets were always used to avoid shearing of worms. Worms were exposed in 24-well tissue-culture treated (TC) plates for 24 hours to DNAN, NTO, or NQ. All test solutions were prepared in K medium except that acetone was used to dissolve DNAN, resulting in 5% carrier in the final solution. Each weel in the TC plates contained 400 worms and 1 ml of K medium or test solution. The nominal concentrations were: Vehicle control, 1.9, 15.5, 62 mg DNA/L, Blank control, 187, 750, 3000 mg NTO/L, and Blank control, 83, 666, 2667 mg NQ/L. Each treatment had 5 replicates.
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Growth protocol |
The wild-type C. elegans var. Bristol, strain N2, was maintained at 20°C on NGM agar plates seeded with Escherichia coli OP50 bacteria. The culture was synchronized at the first larval stage (L1) by bleaching with a 5% sodium hypochlorite solution. Worms were harvested by gentle rinsing with K Medium in late L3 stage which were determined by both length and age (Donkin and Williams 1995; Altun and Hall 2005).
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Extracted molecule |
total RNA |
Extraction protocol |
After the 24-hr exposure, all 400 worms per well were collected into a cryogenic vial and spun at 1150 x g for 2.5 min. Supernatant was removed and the vial were flash frozen in liquid nitrogen and stored at -80°C. Total RNA was extracted from each vial using RNeasy mini kits (Qiagen, Valencia, CA) and treated as one biological replicate. Four of the five replicates (total RNA samples) per treatment were chosen for further microarray hybridization.
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Label |
Cy3
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Label protocol |
One hundred ng of total RNA was first reverse-transcribed into cDNA, followed by cDNA labeling using Low Input Quick Amp Labeling Kits (Agilent, Palo Alto, CA) in the presence of cyanine 3-CTP dye.
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Hybridization protocol |
The labeled cDNA was hybridized to the C. elegans-specific 44K-olig array (one sample/array) at 65°C for 17 hrs using Agilent’s Gene Expression Hybridization Kits. A total of 48 samples (3 chemicals × 4 treatments per chemical × 4 replicates per treatment) were hybridized to six 8 × 44K-array slides in a randomized fashion to reduce systematic errors.
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Scan protocol |
An Agilent high-resolution DNA Microarray Scanner Model G2565CA was used to scan microarray images (resolution = 3 microns per pixel).
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Description |
Labeled cRNA was hybridized to the genome-wide microarray to profile global gene expression.
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Data processing |
Raw microarray gene expression data were extracted from acquired array images as spot and background signal intensity using Agilent’s Feature Extraction software v10.7. A spot was flagged out if its raw signal intensity was below its background level, or if it was saturated. A gene was flagged out if over 50% of the 48 samples had missing values for this gene. The filtered data was background subtracted and log-transformed. The spike-in RNA mix with known composition and RNA copy numbers was used to construct standard linear regression curves, from which the unknown worm RNA concentrations were derived. The derived RNA concentrations were normalized to the median value per array (sample).
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Submission date |
Dec 13, 2016 |
Last update date |
Jun 01, 2017 |
Contact name |
Ping Gong |
E-mail(s) |
ping.gong@usace.army.mil
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Phone |
(601) 634-3521
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Organization name |
U.S Army Engineer Research and Development Center
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Department |
Environmental Laboratory
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Street address |
3909 Halls Ferry Road
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City |
Vicksburg |
State/province |
Mississippi |
ZIP/Postal code |
39180 |
Country |
USA |
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Platform ID |
GPL22795 |
Series (1) |
GSE92365 |
Comparative toxicogenomic effects of three IMX-101 constituents DNAN, NTO and NQ on C. elegans |
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