RNA was collected from renal tissues and purified by an RNeasy kit according to the manufacturer's instructions (Qiagen, Valencia, CA), and real-time PCR was performed with Sybergreen on an Opticon real-time PCR machine (MJ Research,Waltham, MA) as previously described [43 (link), 44 (link)]. Primers used for detection of mRNA expression of KIM-1, MIF, collagen I, α-SMA, TGF-β1, MCP-1, TNFα, and GAPDH were described previously [27 (link), 45 (link)]. GAPDH was used as an internal standard. The ratio for the mRNA was examined against GAPDH and was expressed as mean ± SE.
Opticon real time pcr machine
Manufactured by Bio-Rad
Sourced in United States
The Opticon real-time PCR machine is a laboratory instrument used for quantitative polymerase chain reaction (qPCR) analysis. It is designed to accurately measure and analyze DNA amplification in real-time during the PCR process. The Opticon machine provides precise temperature control and sensitive fluorescence detection to enable reliable quantification of nucleic acid samples.
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Lab products found in correlation
7 protocols using opticon real time pcr machine
1
Renal Tissue RNA Analysis: Real-time PCR Quantification
2
Renal RNA Extraction and qPCR Analysis
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RNA was collected from renal tissues and purified by an RNeasy kit according to the manufacturer's instructions (Qiagen, Valencia, CA), and real-time PCR was performed with Sybergreen on an Opticon real-time PCR machine (MJ Research, Waltham, MA) as previously described. [28 (link)-30 (link)] Primers used for detection of mRNA expression of collagen I, α-SMA, TGF-β1, MCP-1, TNFα, and GAPDH were described previously [28 (link)-30 (link)]. House keep gene GAPDH was used as an internal standard. The ratio for the mRNA was examined against GAPDH and was expressed as mean±SE.
3
Quantification of KALRN Isoforms by qPCR
4
Quantification of Gene Expression Levels
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The remaining three rats in each group were validated using real-time PCR. Spinal cord tissues were separated under anesthetic after the behavioral test. The first-strand cDNA was synthesized using reverse transcription kit (Vazyme) according to the manufacturer's instruction and amplified in triplicate using IQ SYBR green SuperMix reagent (Bio-Rad, Hercules, CA) with an Opticon real-time PCR machine (MJ Research, Waltham, MA), according to the manufacturer's instructions. The specificity of real-time PCR was confirmed via routine agarose gel electrophoresis and melting-curve analysis. The sequences of all primers are shown in Table 5 . The comparative Ct method (ΔΔCt) was used to quantify gene expression, and the relative quantification was calculated as 2−ΔΔCt. Calculation of lncRNAs and circRNAs expression were consistent with mRNA, the internal control were β-actin. The expression levels of miRNAs were normalized to U6 level in each sample.
5
Gene Expression Analysis by RT-qPCR
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Gene expression was analysed by real-time polymerase chain reaction (PCR). To analyse the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), total RNA from the cells in Regions ECA and CS was extracted by Trizol isolation, and cDNA was synthesized using the TaqMan reverse transcription (RT) Master Mix kit according to the manufacturer’s protocol. Real-time PCR analysis was performed with an Opticon Real-Time PCR Machine (Bio-Rad, CA, USA) using the SYBR Green PCR Master Mix Reagent Kit (Takara Biotechnology, Dalian, China). Each reaction mixture (total volume 20 μL) contained cDNA (equivalent to 100 ng RNA), 200 nM deoxyribonucleotides, each primer at 800 nM and 0.5 U GoTaq polymerase (Biotools B&M Labs, Madrid, Spain). The cycling conditions were as follows: incubation for 3 min at 95 °C followed by 35 cycles of 95 °C for 30 s, 55 °C for 45 s and 72 °C for 45 s.
The relative quantitation of the gene expression was determined by the ΔΔCT method, and GAPDH was used as a control. For these analyses, we used the specific primers shown in ST 1.
The relative quantitation of the gene expression was determined by the ΔΔCT method, and GAPDH was used as a control. For these analyses, we used the specific primers shown in ST 1.
6
RNA Extraction and Real-Time PCR Analysis
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Total RNA was extracted from renal tissues and cultured cells and purified using an RNeasy kit according to the manufacturer's instructions (Qiagen, Valencia, CA, USA), and real-time PCR was performed with an Opticon real-time PCR machine (Opticon 2, Bio-Rad Labs., Hercules, CA, USA) using the IQ SYBR Green Supermix reagent (Bio-Rad Labs., Hercules, CA, USA) as previously described (You et al., 2016 (link)). The primer sequences used in the present study are listed in Table 1 .
7
Quantitative PCR Analysis of Neurotransmitter Receptors
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