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Status |
Public on Jul 01, 2024 |
Title |
mitfa_24hpf_rep1 |
Sample type |
RNA |
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Source name |
zebrafish mitfa:GFP positive cells
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Organism |
Danio rerio |
Characteristics |
cell type: mitfa:GFP pigment lineage cells developmental stage: 24hpf
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Treatment protocol |
No treatment
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Growth protocol |
Zebrafish were bred, raised and maintained at 28.5 °C according to standard protocols (Westerfield M, 2000) and were housed at the CSIR-Institute of Genomics and Integrative Biology (IGIB), Mathura Road New Delhi, India. Embryos were staged both using timing (hours post fertilization (hpf); days post fertilization (dpf)) and morphological features according to (Kimmel, 1995)
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was isolated from each of these sorted populations according to manufacturer’s protocols (Nucleospin RNA XS kit; MachereyNagel)
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Label |
Cy3
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Label protocol |
The microarray hybridization and scanning were performed at the Agilent certified microarray facility of Genotypic Technology, Bengaluru, India. The samples for Gene expression were labeled using Agilent Quick-Amp labeling Kit (p/n5190-0442). Synthesized double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up using Qiagen RNeasy columns (Qiagen, Cat No: 74106) and quality assessed for yields and specific activity using the Nanodrop ND-1000
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Hybridization protocol |
Labeled cRNA sample were fragmented at 60oC and hybridized on to Agilent Zebrafish Gene Expression Microarray 8X60K. Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of (Agilent Technologies, In situ Hybridization kit, Part Number 5190-0404). Hybridization was carried out in Agilent’s Surehyb Chambers at 65o C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Agilent Technologies, Part Number 5188-5327)
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Scan protocol |
The hybridized slides were scanned using the Agilent Microarray Scanner (Agilent Technologies, Part Number G2600D). Raw data extraction from Images was obtained using Agilent Feature Extraction software
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Data processing |
Images were quantified using Feature Extraction Software (Version-11.5 Agilent). Feature extracted raw data was analyzed using GeneSpring GX software from Agilent. Normalization of the data was done in GeneSpring GX using the 75th percentile shift (Percentile shift normalization is a global normalization, where the locations of all the spot intensities in an array are adjusted. [This normalization takes each column in an experiment independently, and computes the percentile of the expression values for this array, across all spots (where n has a range from 0-100 and n=75 is the median). It subtracts this value from the expression value of each entity.
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Submission date |
Nov 17, 2021 |
Last update date |
Jul 01, 2024 |
Contact name |
Vivek T Natarajan |
E-mail(s) |
tnvivek@igib.in
|
Organization name |
CSIR-IGIB
|
Lab |
Pigment Cell Biology Lab
|
Street address |
Mathura Road
|
City |
Delhi |
State/province |
Delhi |
ZIP/Postal code |
110025 |
Country |
India |
|
|
Platform ID |
GPL26813 |
Series (1) |
GSE189059 |
Time-course gene expression analysis of melanocyte development |
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