Hearts were snap-frozen in liquid nitrogen, and pulverized and immersed in Trizol (Molecular Research Center, TR118). Total RNA was isolated, and reverse-transcribed using iScript cDNA synthesis kit (Bio-Rad). Quantitative PCR was performed using Brilliant II SYBR Green (Agilent Technologies) (Schwaerzer et al., 2019 (link)). All primers were intron-spanning (except for 18S rRNA), and were tested with serial cDNA dilutions. Relative changes in mRNA expression were analyzed using the comparative 2−ΔΔCt method, with 18S rRNA as internal control.
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Quantitative Analysis of mRNA Expression
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Corresponding Organization : University of California, San Diego
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Variable analysis
independent variables
- None explicitly mentioned
- Relative changes in mRNA expression
- Tissue type (hearts)
- RNA isolation method (Trizol)
- Reverse transcription method (iScript cDNA synthesis kit)
- Quantitative PCR method (Brilliant II SYBR Green)
- Internal control (18S rRNA)
- Positive control: Serial cDNA dilutions to test primers
- Negative control: Not explicitly mentioned
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