Total RNA was extracted using RNeasy mini kit (Qiagen, Antwerp, Belgium) according to the manufacturer’s protocol. qRT-PCR reactions were performed in triplicates, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as housekeeping gene in a mini-Opticon Instrument (Bio-Rad, Temse, Belgium) with 200 nM of each primer. Results were analyzed using MJ Opticon Monitor Analysis software Version 3.1 (Bio-Rad). Primer sequences were: Sox2 fw: 5′-TGCTGCCTCTTTAAGACTAGGG-3′ Sox2 rev: 5′-TCGGGCTCCAAACTTCTC-3′; Sox17 fw: 5′-CTTTATGGTGTGGGCCAAAG-3′ Sox17 rev: 5′-TTGTAGTTGGGGTGGTCCTG-3′ Cx40 fw: 5′-AGCAGCCAGAGCCTGAAGAA-3′ Cx40 rev: 5′- CAGGACAGTGAGCCAGACCT-3′. Primers were synthesized by PRIMM, Milan, Italy. Extra-cellular matrix (ECM), adhesion and ECM gene expression was analyzed by RT2 Profiler™ PCR Arrays from Sabioscience (Qiagen, Antwerp, Belgium) following manufacturer’s instructions.
Miniopticon instrument
Manufactured by Bio-Rad
Sourced in Belgium, United States
The MiniOpticon is a real-time PCR detection system designed for DNA amplification and detection. It features a compact design and a four-channel optical system that can detect up to four different fluorescent dyes simultaneously. The MiniOpticon is capable of performing quantitative and qualitative PCR analyses.
Automatically generated - may contain errors
Lab products found in correlation
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Rneasy plus micro kit, by Qiagen (2 mentions)
Quantitect sybr green pcr kit, by Qiagen (2 mentions)
Mj opticon monitor analysis software version 3, by Bio-Rad (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
4 protocols using miniopticon instrument
1
Quantification of Stem Cell Markers
2
Quantifying Unilateral Naris Occlusion in OE
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cDNA samples for qPCR analysis were prepared using the QuantiTect Reverse Transcription Kit (QIAGEN) starting from RNA prepared from dissociated single OE cells (150 μL) using the RNeasy Plus Micro Kit (QIAGEN). qPCR experiments were performed using the QuantiTect SYBR Green PCR Kit (QIAGEN) with a MiniOpticon instrument (Bio-Rad). Primer pairs for S100a5 and Omp (Table S2 ) were designed using the Primer-BLAST tool (NCBI), synthesized by Integrated DNA Technologies, and used to quantify S100a5 and Omp levels within cDNA pairs corresponding to the open and closed sides of an OE. For each sample, the ratio of S100a5 (activity-dependent expression) to Omp (activity-independent expression) was calculated. UNO was considered successful for OE sample pairs exhibiting an S100a5/Omp ratio at least 5-fold higher in the open sample compared to the closed sample.
3
Quantitative Real-Time PCR Analysis
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Total RNA was extracted using Trizol® Reagent (Life Technologies) according to the manufacturer's instructions. cDNA was generated using the PrimeScript™ RT reagent kit (Takara, Otsu, Japan). The sequences of forward and reverse primers are listed in Table 1 . All PCRs were carried out using SYBR® Premix Ex Taq™ II (Takara) and Mini Opticon instrument (BioRad, Hercules, CA, USA). The real-time cycling conditions were as follows: initial enzyme activation at 50℃ for 2 min and denaturation at 95℃ for 10 min followed by 40 cycles of denaturation at 95℃ for 15 sec and annealing/extension at 60℃ for 1 min. The product purity was confirmed by a dissociation curve analysis. The mRNA levels of the target genes were normalized to the values of β-actin, and presented as fold changes relative to controls (no arctiin treatment).
4
Quantifying Unilateral Naris Occlusion in OE
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