|
| Status |
Public on Jan 02, 2013 |
| Title |
PA (array) |
| Sample type |
RNA |
| |
|
| Source name |
PA liver, brain and muscle
|
| Organism |
Squalius alburnoides |
| Characteristics |
genome composition: PA
tissue: liver, brain, muscle
developmental stage: adult
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Fishes were sacrificed by overdose on anaesthetic MS222, and the respective tissues/organs were dissected and preserved in RNAlatter® (Ambion) at -20ºC. Total RNA was extracted from liver, muscle and brain using the Tri-Reagent® (Ambion) following the supplier's instructions. Contaminant DNA was eliminated by the addition of TURBO™ DNase (Ambion) followed by purification with phenol/chloroform. Ethanol, Glycogen and Sodium Acetate (NaOAc) were used to achieve RNA precipitation. 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 all samples was labeled using the ULS microRNA labeling kit (Kreatech) according to the manufacturer’s instructions. Briefly, 2 ug of total RNA from each sample 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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| |
|
| 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. Warm the prehybridization solution to 50 ºC in a 100 ml Coplin Jar. 3. Place slides to be analyzed into the pre-warmed 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.
|
| Scan protocol |
Slides were scanned using the Agilent G2565AA DNA microarray scanner: Resolution: 10, Red laser PMT= 0, Green laser PMT= 100.
|
| 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 200% background. A global median normalization of miRNA microarray data was applied using BRB-ArrayTools v3.4.0 software. Cy3 intensities were obtained.
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| |
|
| Submission date |
Mar 28, 2012 |
| Last update date |
Jan 02, 2013 |
| Contact name |
Angela Inácio |
| E-mail(s) |
mainacio@fc.ul.pt
|
| Organization name |
centro de Biologia Ambiental
|
| Street address |
Campo Grande
|
| City |
Lisbon |
| ZIP/Postal code |
1749-16 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL14672 |
| Series (2) |
| GSE36894 |
miRNA expression profile in the Squalius alburnoides complex |
| GSE38799 |
miRNA expression profile and small RNA transcriptome in the Squalius alburnoides complex |
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