mRNA expression of vastus lateralis was conducted as follows [34 (link)]. Total RNA was isolated by homogenizing 10–15 mg tissue with a hand-held homogenizer in a solution containing Tri reagent LS (Molecular Research Centre, Cincinnati, OH, USA). The RNA was separated and precipitated using chloroform and isopropanol. Extracted RNA was washed with ethanol then suspended in nuclease-free water with EDTA. RNA concentration was determined using the EPOCH (Take3; BioTek) spectrophotometer. cDNA was synthesized using a commercially available kit (iScript, BioRad, Hercules, CA, USA). All isolated RNA and cDNA samples were stored at −80°C until analysis. Real-time PCR was carried out with a CFX Connect real-time PCR cycler (BioRad) under similar protocol as reported previously [35 (link), 36 ] using SYBR green custom designed primers for beta 2-microglobulin (β2M), cluster of differentiation 68 (CD68), cluster of differentiation 45 (CD45), interleukin 6 (IL-6) and C-C motif chemokine ligand 2 (CCL2) which have been previously described [37 (link)] in addition to myoD and myogenin [38 (link)]. Cycle threshold values of target genes were normalized to β2M then fold change values were calculated (ΔΔCt). β2M remained stable across the interventions.
Protocol detail
Vastus Lateralis mRNA Expression Analysis
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Beta 2 microglobulin
C c motif chemokine 2
Cdna
Chloroform
Edta
Epoch
Ethanol
Genes
Held
Interleukin 6
Isopropanol
Ligand
Mrna
Myogenin
Primers
Real time pcr
Sybr green
Tissue
Vastus lateralis
Corresponding Organization : University of Utah
Top 5 similar protocols
Variable analysis
independent variables
- MRNA expression of genes
- MRNA expression levels of beta 2-microglobulin (β2M), cluster of differentiation 68 (CD68), cluster of differentiation 45 (CD45), interleukin 6 (IL-6), C-C motif chemokine ligand 2 (CCL2), myoD and myogenin
- Tissue sample size (10-15 mg)
- Hand-held homogenizer used for tissue homogenization
- Tri reagent LS used for RNA isolation
- Chloroform and isopropanol used for RNA separation and precipitation
- Ethanol used for RNA washing
- EDTA used in nuclease-free water for RNA suspension
- EPOCH (Take3; BioTek) spectrophotometer used for RNA concentration determination
- IScript (BioRad, Hercules, CA, USA) kit used for cDNA synthesis
- CFX Connect real-time PCR cycler (BioRad) used for real-time PCR
- SYBR green custom designed primers used for target genes
- β2M remained stable across the interventions
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