In the assessment of mRNA transcripts of specific genes in skeletal muscle, RNA was isolated with TRIzol® reagent (Life Technologies, Carlsbad, CA, USA) according to the instructions of the manufacturer. The RNA quantification and purity was assessed by nanodrop-1000 (Thermo Scientific, Walthan, MA, USA). Aliquots (2µg) of total RNA were reverse-transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the protocol of the manufacturer. Quantitative PCR (q-PCR) was performed for different genes (TFAM, Myh4, SOD-1, SOD-2), in a final reaction volume of 20 µl containing 10 µl PerfeCTa SYBR Green SuperMix, Low ROX (Quanta Biosciences, Gaithersburg, MD), 6 µl nuclease free water, 2µl cDNA, 40 picomoles of forward, and reverse primers. Table 1 presents all sequence-specific oligonucleotide primers (Invitrogen, Carlsbad, CA, USA). The data was represented in fold expression. This was calculated as the cycle threshold difference between control and sample normalized with the housekeeping genes beta actin and 18s.
Sequence specific oligonucleotide primers
Manufactured by Thermo Fisher Scientific
Sourced in United States
Sequence-specific oligonucleotide primers are short, synthetic DNA molecules designed to serve as starting points for DNA amplification processes, such as Polymerase Chain Reaction (PCR). These primers are designed to hybridize to a specific target sequence within a DNA or RNA template, allowing for the selective and efficient amplification of the desired genetic material.
Automatically generated - may contain errors
2 protocols using sequence specific oligonucleotide primers
1
Quantitative Analysis of mRNA Transcripts in Skeletal Muscle
2
Triterpenoid Effects on Inflammatory Markers
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Adipocytes, myotubes and macrophages were treated at different concentrations of the triterpenoid mixtures according to the cell type (3.13, 6.25, 12.5, 25, 50, 100 and 200 The expression of the inflammatory marker genes TNF-α, IL-1β and IL-6 was evaluated and the relative amount of the whole mRNA was calculated using the comparative or ΔΔCt method. All the sequencespecific oligonucleotide primers (Table SIV) were obtained from Invitrogen. Serial cDNA dilution curves were produced in order to calculate the amplification efficiency of all genes. A graph of threshold cycle (Ct) vs. log10 relative copy number of the sample from a dilution series was produced. The slope of the curve was used to determine amplification efficiency using the formula: Efficiency (%) = (10^(-1/The Slope Value)-1)*100.
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