Real-time PCR was run on a qTOWER3-thermocycler (Analytik Jena AG, Jena, Germany). Each reaction mixture of 20 µL contained 10 µL of innuMIX qPCR MasterMix Probe (Analytik Jena AG, Jena, Germany), 3.8 µL of distilled water, 0.5 µL of forward primer (20 µM), 0.5 µL of reverse primer (20 µM), 0.2 µL of probe (20 µM), and 5 µL of DNA template (10 ng/µL). The thermocycling conditions were set as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s, and 60 °C for 1 min.
Innumix qpcr master mix probe
Manufactured by Analytik Jena
Sourced in Germany
The InnuMix qPCR Master Mix Probe is a ready-to-use solution for quantitative real-time PCR (qPCR) experiments using hydrolysis probe detection. It contains all the necessary components, including a proprietary DNA polymerase, for efficient and sensitive amplification of DNA targets.
Automatically generated - may contain errors
3 protocols using innumix qpcr master mix probe
1
Real-time PCR Quantification Protocol
2
Quantification of Myosin Heavy Chain Isoforms by qRT-PCR
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The mRNA quantification of four different myosin heavy chain isoforms (Myh1, Myh2, Myh4, and Myh7) in the concerning muscle tissues was performed by qRT-PCR (Taq-Man® Assays; PE Applied Biosystems®, Weiterstadt, Germany) in comparison to 18S rRNA (Eucaryotic 18S rRNA Endogenous control: 4310893E; PE Applied Biosystems®, Weiterstadt, Germany) using gene-specific TaqMan PCR probes and primers which have been previously described elsewhere [60 (link)]. The master mix contained innuMix qPCR Master Mix Probe (Analytik Jena AG), 10x specific probes and primers, and RNase free water. For quantification of gene expression of the different MyHCs, 8 ng cDNA was used in a final volume of 20 μL. Gene amplification was performed with the TOptical cycler (Analytik Jena AG) at 95°C for 10 minutes for initial denaturation followed by 40 cycles, in each case 10 seconds at 95°C and 45 seconds at 60°C. Absolute copy numbers of the studied genes and 18S cDNA were determined using calibration curves generated with cloned PCR fragment standards [60 (link)]. Copy numbers of individual transcripts were given in relation to those of 18S cDNA. Each probe was performed twice and a “nontemplate control” was carried out parallel in all experiments to validate results.
3
Quantifying Gene Expression in Rat Muscle
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Homogenization of the muscle samples and total RNA isolation were performed using TRIzol and QIAzol lysis reagents (Qiagen Inc., Hilden, Germany) and the RNeasy ® Lipid Kit (Qiagen) according to manufacturer's instructions. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) for cDNA synthesis was done in the Thermocycler TOptical Real-Time PCR (Analytik Jena AG, Jena, Germany) using 200 ng RNA and innuSCRIPT Reverse Transcriptase, innuNucleotide Mix and Random primer (Analytik Jena AG).
The following genes were quantified: collagen type 1 and type 2 (COL1A1 and COL2A1); insulin-like growth factor (IGF) 1 and IGF2; vascular endothelial growth factor (VEGF); and myostatin (MSTN), a negative regulator of muscle growth. The quantification of the expression of different rat genes were performed as described previously by using the master mix contained in the innuMix qPCR Master Mix Probe (Analytik Jena AG), ×10 specific probes and primers (IGF1: Rn 00710306_m1; IGF2: Rn 00580426_m1; VEGF: Rn 01511602_m1; MSTN: Rn 00569683_m1; COL1A1: Rn 01463848_m1; COL2A1: Rn 00563954_m1; Eukaryotic 18S rRNA Endogenous Control: 4310893E; PE (Applied Biosystems, Weiterstadt, Germany) and RNase free water. 7, 27 Fig. 1. Surgical approach: A -skin incision above the latissimus dorsi; B -insertion of the oxidized cellulose; C -wound closure using skin staples
The following genes were quantified: collagen type 1 and type 2 (COL1A1 and COL2A1); insulin-like growth factor (IGF) 1 and IGF2; vascular endothelial growth factor (VEGF); and myostatin (MSTN), a negative regulator of muscle growth. The quantification of the expression of different rat genes were performed as described previously by using the master mix contained in the innuMix qPCR Master Mix Probe (Analytik Jena AG), ×10 specific probes and primers (IGF1: Rn 00710306_m1; IGF2: Rn 00580426_m1; VEGF: Rn 01511602_m1; MSTN: Rn 00569683_m1; COL1A1: Rn 01463848_m1; COL2A1: Rn 00563954_m1; Eukaryotic 18S rRNA Endogenous Control: 4310893E; PE (Applied Biosystems, Weiterstadt, Germany) and RNase free water. 7, 27 Fig. 1. Surgical approach: A -skin incision above the latissimus dorsi; B -insertion of the oxidized cellulose; C -wound closure using skin staples
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