|
| Status |
Public on Aug 08, 2012 |
| Title |
Day 4, rep 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Liver tissue, Control, Day 4 post infection
|
| Organism |
Lates calcarifer |
| Characteristics |
tissue: liver
developmental stage: juvenile
condition: control
time post-infection: day 4
|
| Treatment protocol |
Fish were screened for ectoparasites via body and gill smears, acclimatized for 2 weeks and later randomly separated into infected and control groups. Every fish in the infected group was challenged with 4,000-8,000 therons individually.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Snaped frozen liver's tissue were homogenized with mortar and pestle, and total RNA was extracted using TRIREAGENT® (Molecular Research Center, USA) following the manufacturer’s instructions. The quality and quantity of the total RNA were determinate by NanoDrop Spectrophotometer ND-1000 (Nanodrop Technologies, USA) and Agilent 2100 Bioanalyzer (Agilent, USA).
|
| Label |
Cy3
|
| Label protocol |
10 μg total RNA were converted to cDNA and labeled with either a Cy3 or Cy5 dye using the Post labelling CyscribeTM kit (Amersham Biosciences, USA) as per manufacturerss instructions. Samples were purified using the CyScribeTM GFXTM purification kit (Amersham Biosciences, USA) before hybridization.
|
| |
|
| Channel 2 |
| Source name |
Liver tissue, Infected, Day 4 post infection
|
| Organism |
Lates calcarifer |
| Characteristics |
tissue: liver
developmental stage: juvenile
condition: infected
time post-infection: day 4
|
| Treatment protocol |
Fish were screened for ectoparasites via body and gill smears, acclimatized for 2 weeks and later randomly separated into infected and control groups. Every fish in the infected group was challenged with 4,000-8,000 therons individually.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Snaped frozen liver's tissue were homogenized with mortar and pestle, and total RNA was extracted using TRIREAGENT® (Molecular Research Center, USA) following the manufacturer’s instructions. The quality and quantity of the total RNA were determinate by NanoDrop Spectrophotometer ND-1000 (Nanodrop Technologies, USA) and Agilent 2100 Bioanalyzer (Agilent, USA).
|
| Label |
Cy5
|
| Label protocol |
10 μg total RNA were converted to cDNA and labeled with either a Cy3 or Cy5 dye using the Post labelling CyscribeTM kit (Amersham Biosciences, USA) as per manufacturerss instructions. Samples were purified using the CyScribeTM GFXTM purification kit (Amersham Biosciences, USA) before hybridization.
|
| |
|
| |
| Hybridization protocol |
Prehybridization of slides was according to Hegde et al. (2000). A Concise Guide to cDNA Microarray Analysis-II. Biotechniques 29(3):548-562. Labelled cDNA were hybridized to the L. calcarifer array and put inside an incubation chamber (Genetix, UK). The incubation chamber was placed in a water bath for 18 h at 42 °C. Arrays then were washed according to Corning® GAPS II slide (USA) instructions.
|
| Scan protocol |
Arrays were scanned immediately with a GenePix 4100A (Axon Instruments, USA) scanner, software GenePixTM Pro 6.0.
|
| Description |
Biological replicate 2 of 3 day 4 post infection.
|
| Data processing |
The GenePix results (.gpr) file was further analyzed by GeneSpring GX 7.3.1, which applied intensity-dependent LOWESS normalization to the results and calculated normalized Cy5 to Cy3 ratios.
|
| |
|
| Submission date |
Dec 27, 2011 |
| Last update date |
Aug 08, 2012 |
| Contact name |
Abdul Munir Abdul Murad |
| E-mail(s) |
munir8488@gmail.com, munir@pkrisc.cc.ukm.my
|
| Organization name |
Universiti Kebangsaan Malaysia
|
| Department |
Faculty of Science & Technology
|
| Lab |
School of Bioscience & BioTechnology
|
| Street address |
Jalan Universiti
|
| City |
Bangi |
| State/province |
Selangor |
| ZIP/Postal code |
43600 |
| Country |
Malaysia |
| |
|
| Platform ID |
GPL15067 |
| Series (1) |
| GSE34745 |
Transcriptome Analysis of the Lates calcarifer Immune Response Towards Cryptocaryon irritans Infection |
|
