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| Status |
Public on Jun 18, 2012 |
| Title |
Embryos_EtOH1.5%_24hpf_R2 |
| Sample type |
RNA |
| |
|
| Source name |
24 hours post fertilization tissues: embryos in EtOH 1.5% solution
|
| Organism |
Danio rerio |
| Characteristics |
age: 24hpf
tissues: embryos
treatment: 1.5% EtOH
|
| Treatment protocol |
Zebrafish embryos were allowed to grow in system water or in 1% or 1.5% ethanol solutions from 4hpf until 24hpf.
|
| Growth protocol |
Zebrafish embryos were collected and Petri dishes with approximately 250 eggs each were incubated at 28ºC to allow normal zebrafish development until 4hpf, when blastula is reached. At this stage, embryos were examined under a dissecting microscope and those that had developed normally were selected for EtOH exposure (approximately 200 eggs). Briefly, 200 embryos were randomly distributed into plastic Petri dishes containing 20 mL of EtOH test solutions (1% EtOH, 1.5% EtOH) or system water for control conditions. All solutions were made by dilution of absolute EtOH in system water. Exposure was from 4hpf to 24hpf.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 24hpf samples using TRIzol® (Invitrogen) according to the manufacturer's instructions and treated with DNaseI (Invitrogen). RNA quantity and quality was assessed using the Nanodrop and Agilent 2100 bioanalyzer systems, respectively. Samples with a RIN number above 7 were used.
|
| Label |
Cy3
|
| Label protocol |
Total RNA from control and test samples were labeled using the ULS microRNA labeling kit (Kreatech) according to the manufacturer’s instructions. Briefly, 2 ug of total RNA from control and test samples were incubated with Cy3-ULS for 15 min at 85ºC. The labeled RNAs were purified to remove non-reacted Cy-ULS to produce a fluorescently-labeled RNA sample for microarray analysis. Dye incorporation was monitored by UV-visible spectroscopy.
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| |
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| Hybridization protocol |
PREHYBRIDIZATION: 1. Make 100 ml prehybridization buffer (for a maximum of 4 slides) containing 1M Tris-HCL pH=9 pre-heated at 50ºC, 100mM ethanolamide and 0.1% SDS. 2. Heat the prehybridization solution to 50 ºC in a 100 ml Coplin Jar. 3. Place slides to be analyzed into the pre-heated pre-hybridization buffer. Incubate for 20 minutes at 50 ºC with constant agitation, if possible. 4. Wash thoroughly by inverting or gently shaking the container with nuclease-free water constantly for 1 minute. Decant the water and repeat this rising step with fresh nuclease-free water. 5. Allow the slides to dry by centrifuging 3 minute 800 rpm. Slides should be used immediately following prehybridization. HYBRIDIZATION: Pre-heat 3x hybridization solution (Ambion) to 65 ºC for at least 5 min to overcome SDS precipitation. Combine labeled RNA with hybridization buffer (preheated to 65 ºC). Heat at 95 ºC for 3 minutes. Immediately put on ice for at least 1 min. Spin-down briefly. Add 4x blocking reagent (Kreatech). Incubate on dark for 1 minute at room temperature. Using Agilent gasket slides (G2534-60003), Agilent SureHyb hybridization chambers (G2534A) and hybridization oven (G2545A), see Agilent User Manual for instructions: 1. For each array, put a gasket slide onto a SureHyb chamber and add a total of 250 ul target as 1 dot in the center of the chamber. 2. Put a microarray slide on the gasket slide by holding it in place with your fingertips, towards the top edge of the gasket chamber. 3. Gently (!) allow the microarray slide to drop onto the gasket chamber by lowering it with the aid of your other hand. Do not move the microarray slide after it is in place. 4. Place the SureHyb chamber into the hybridization oven at 42 ºC for 16 hours.
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| Scan protocol |
Scanning of slides using the Agilent G2565AA DNA microarray scanner: Resolution: 10, Red laser PMT= 0, Green laser PMT= 100
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| Data processing |
Microarray images were analyzed using Quantarray v3.0 software (PerkinElmer). Manually flagged bad spots were eliminated and the Cy3 median pixel intensity values were background subtracted, followed by the averaging of quadruplicate features on the array. Data points were removed when intensity values were below 100. A global median normalization of zebrafish 24hpf larvae microarray data was applied using BRB-ArrayTools v3.4.0 software. Cy3 intensities were obtained. Data was normalized for each miRNA with the correspondent NCode™ Positive Control probe intensity. Log-transformed (base 2) of the ratio between miRNA and NCode™ Positive Control intensities of each sample was calculated.
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| Submission date |
Oct 05, 2011 |
| Last update date |
Jun 18, 2012 |
| Contact name |
Patrícia Matos Pereira |
| E-mail(s) |
pereirap@ua.pt
|
| Organization name |
University of Aveiro
|
| Department |
Biology
|
| Lab |
RNA biology
|
| Street address |
Campus Universitário de Santiago
|
| City |
Aveiro |
| ZIP/Postal code |
3810-183 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL14672 |
| Series (1) |
| GSE32632 |
Ethanol alters microNA expression in zebrafish embryos |
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