Total RNA was isolated from LV tissue using TRIzol reagent (Invitrogen), and cDNA was synthesized from 1 µg RNA using the iScript™ cDNA Synthesis kit (BioRad). Relative levels of mRNA transcripts for atrial natriuretic factor (ANF), connective tissue growth factor (CTGF), TNF, interleukin(IL)-1β, IL-6, IL-10, matrix metalloproteinase(MMP)-2, and MMP-9 were quantified by real-time PCR using SYBR® Green (Applied Biosystems). Data were normalized to GAPDH expression using the CT comparative method [14 (link)]. Primer pairs are listed in Supplemental Table 1.
Protocol detail
Quantifying Gene Expression in Heart Tissue
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Atrial natriuretic factor
Cdna
Connective tissue growth factor
Gapdh
Il 10
Il 1β
Interleukin
Matrix metalloproteinase
Mmp 2
Mmp 9
Mrna
Primer
Real time pcr
Sybr green
Synthesis
Tissue
Trizol
Corresponding Organization :
Other organizations : University of Louisville
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Variable analysis
independent variables
- None explicitly mentioned
- Relative levels of mRNA transcripts for atrial natriuretic factor (ANF)
- Relative levels of mRNA transcripts for connective tissue growth factor (CTGF)
- Relative levels of mRNA transcripts for TNF
- Relative levels of mRNA transcripts for interleukin(IL)-1β
- Relative levels of mRNA transcripts for IL-6
- Relative levels of mRNA transcripts for IL-10
- Relative levels of mRNA transcripts for matrix metalloproteinase(MMP)-2
- Relative levels of mRNA transcripts for MMP-9
- GAPDH expression
- Positive control: Not mentioned
- Negative control: Not mentioned
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