Total RNA was isolated from individual whole corneas using RNA-STAT 60 (Tel-Test; Friendswood, TX, USA) according to the manufacturer’s recommendations and quantified by spectrophotometric determination (260 nm). Total RNA (100 ng) was reverse transcribed and used to produce a cDNA template. cDNA was amplified using SYBR® Green Master Mix according to the manufacturer. Each 10 μL reaction mixture contained: 5 μL 2× SYBR Green PCR Master Mix, 1 μL DEPC-water, 1 μL forward and reverse primers, 2 μL cDNA (diluted 1:10). All primers for the PCR reaction were designed using Primer3 PCR primer design software and semi-quantitative real-time RT-PCR was carried out using the CFX Connect Real-Time RT-PCR Detection System (BioRad, Hercules, CA). PCR amplification conditions were determined using established methods [25 (link)]. Relative transcript levels were calculated using the relative standard curve method comparing the amount of target (gene of interest) normalized to an endogenous reference (β-actin). Data are shown as the mean fold-change ± SD compared to basal levels of uninfected controls and represent at least three individual experiments.
Cfx connect real time rt pcr detection system
Manufactured by Bio-Rad
Sourced in United States
The CFX Connect Real-Time RT-PCR Detection System is a laboratory instrument designed for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. It is capable of detecting and quantifying specific nucleic acid sequences in samples.
Automatically generated - may contain errors
4 protocols using cfx connect real time rt pcr detection system
1
Quantitative Real-Time RT-PCR of Corneal RNA
2
Quantitative RNA Expression Analysis
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Total RNA was isolated from individual whole corneas at 3 and 5 days p.i. for gene expression analysis using RNA-STAT 60 (Tel-Test, Friendswood, TX, USA) according to the manufacturer’s recommendations and quantified by spectrophotometric determination (260 nm). 100 ng of total RNA was reverse transcribed and used to produce a cDNA template and amplified as previously described [29 (link)]. All primers for the PCR reaction were designed using Primer3 (Table 1 ). Semiquantitative real-time RT-PCR was performed using the CFX Connect Real-Time RT-PCR Detection System (BioRad, Hercules, CA, USA). PCR amplification conditions were determined using routine methods [30 (link)]. Relative transcript levels were determined using the relative standard curve method comparing the amount of target normalized to an endogenous reference, β-actin. Data are shown as the mean ± SD for relative transcript levels normalized to β-actin and relative to the expression of uninfected (normal) controls.
3
RNA Extraction and Real-Time RT-PCR Analysis
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RNA was extracted by RNA STAT-60 (Tel-Test, Friendswood, TX, USA) per the manufacturer's protocol and subjected to real-time RT-PCR analyses. Total RNA extracted for HREC and MIO-M1 was quantitated by spectrophotometric determination (260 nm). Total RNA (100 ng) was reverse transcribed and used to produce a cDNA template as previously described [31 (link)]. cDNA products were diluted 1 : 20 with DEPC-treated water, and 2 μL cDNA (10 μL total reaction volume) was used for semiquantitative real-time RT-RT-PCR analysis (CFX Connect Real-Time RT-PCR Detection System; Bio-Rad, Hercules, CA, USA). All human primer pair sequences designed in the laboratory (PrimerQuest, Integrated DNA Technologies, Coralville, IA, USA) are listed in Table 1 . RT-PCR amplification conditions were determined using routine methods [32 (link)]. Relative transcript levels were calculated using the relative standard curve method comparing the amount of targets normalized to an endogenous reference, β-actin. Data are shown as mean ± SD for relative transcript levels and represent at least two individual experiments.
4
Quantitative Real-Time RT-PCR Analysis of Corneal Gene Expression
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Total RNA was isolated from individual whole corneas for gene expression analysis using RNA-STAT 60 (Tel-Test, Friendswood, TX, USA), according to the manufacturer’s recommendations, and quantified by spectrophotometric determination (260 nm). cDNA templates were constructed by reverse transcribing 100 ng of total RNA, then amplified using SYBR® Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), per the manufacturer’s instruction, with the reaction mixture previously described [49 (link)]. All primers were generated using Primer3 PCR v. 4.1.0 primer design software, and primer sequences are shown in Table 1 . Semi-quantitative real-time RT-PCR was carried out using the CFX Connect Real-Time RT-PCR Detection System (BioRad, Hercules, CA, USA). Changes in mRNA expression were calculated using the relative standard curve method comparing the amount of target normalized to an endogenous reference, β-actin [50 (link)]. Results are reported as the mean fold change ± SD normalized to β-actin and relative to the expression of uninfected (normal) corneas.
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