Total RNA was isolated from whole larvae at 72 (day 3), 96 (day 4), and 120 (day 5) hpf using RNAzol RT (Molecular Research Centre, Cincinnati, OH). Each RNA sample was a composite of approximately 20–30 larvae. RNA was converted to cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription kit (Life Technologies, Carlsbad, CA), and qPCR for target genes was performed using Power SYBR Green PCR Master Mix with a 7500 Fast Real-Time (Applied Biosystems, Foster City, CA). Primers were purchased from Integrated DNA Technologies (Coralville, IA) as described previously (Cocco et al., 2017 (link); Manchanda et al., 2019 (link)). Relative gene expression was quantified using the ΔΔCt method using β-actin as a housekeeping gene for normalization (Pfaffl, 2001 (link)).
Protocol detail
Temporal Gene Expression Analysis in Zebrafish Larvae
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Applied Biosystems High-Capacity cDNA Reverse Transcription kit
Power SYBR Green PCR Master Mix
7500 Fast Real-Time
Actin
Cdna
Gene expression
Genes
Housekeeping gene
Larvae
Primers
Real time pcr
Reverse transcription
Sybr green
Corresponding Organization : Oregon State University
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Variable analysis
independent variables
- Time points (72 hpf, 96 hpf, 120 hpf)
- Relative gene expression of target genes
- Approximately 20-30 larvae per RNA sample
- β-actin as a housekeeping gene for normalization
- No positive or negative controls were explicitly mentioned in the information provided.
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