Total RNA was extracted from cells with TRIzol reagent (Invitrogen), and 500 ng RNA was reverse-transcribed with PrimeScript RT reagent kit (TaKaRa). qPCR was performed with SYBR green reagent (Bio-Rad) in the MX3000 qPCR system (Agilent). The primers used for qPCR are listed in Table S2 .
Mx3000 qpcr system
Manufactured by Agilent Technologies
Sourced in United States
The Mx3000P QPCR System is a real-time PCR instrument designed for quantitative analysis of DNA and RNA samples. It features a compact design, a high-resolution optical system, and an intuitive software interface. The system is capable of performing quantitative real-time PCR experiments, gene expression analysis, and other genomic applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr green reagent, by Bio-Rad (1 mentions)
Tb green premix ex taq 2 kit, by Takara Bio (1 mentions)
Primerscript rt master mix, by Takara Bio (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Superscript 2 rt kit, by Thermo Fisher Scientific (1 mentions)
Rnase h, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Agilent Technologies (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Revertaid h minus reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using mx3000 qpcr system
1
Quantitative RNA Expression Analysis
2
Quantifying RNA Expression in Skin Cells
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Total RNA was extracted using PureLink RNA Mini Kit (ThermoFisher Scientific, Waltham, MA, USA). Total RNA concentration was measured using a NanoDrop micro-spectrophotometer, and the aliquots were stored at −80 °C until use. Total RNA was reverse-transcribed to cDNA using PrimerScript RT Master Mix (Takara Bio, Shiga, Japan).
Real-time PCR was conducted using a TB Green Premix ExTaq II kit (Takara Bio, San Jose, CA, USA) and a Mx3000 QPCR System (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. The primer pair for involucrin was: forward (5′-GGTCCAAGACATTCAACCAGCC-3′) and reverse (5′-TCTGGACACTGCGGGTGGTTAT-3′), CerS3 forward (5′-CATGATCTTGCAGGTCCTTCACC-3′) and reverse (5′-CTCGTCATCACTCCTCACATCC-3′), and GAPDH forward (5′- GTCTCCTCTGACTTCAACAGCG-3′) and reverse (5′- ACCACCCTGTTGCTGTAGCCAA-3′). The PCR reaction conditions were as follows: 1 cycle at 95 °C for 10 s, then 40 cycles at 95 °C for 5 s, at 60 °C for 30 s, at 95 °C for 1 min, at 60 °C for 30 s, and at 95 °C for 30 s. Relative mRNA levels of involucrin and CerS3 were normalized by GAPDH.
Real-time PCR was conducted using a TB Green Premix ExTaq II kit (Takara Bio, San Jose, CA, USA) and a Mx3000 QPCR System (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. The primer pair for involucrin was: forward (5′-GGTCCAAGACATTCAACCAGCC-3′) and reverse (5′-TCTGGACACTGCGGGTGGTTAT-3′), CerS3 forward (5′-CATGATCTTGCAGGTCCTTCACC-3′) and reverse (5′-CTCGTCATCACTCCTCACATCC-3′), and GAPDH forward (5′- GTCTCCTCTGACTTCAACAGCG-3′) and reverse (5′- ACCACCCTGTTGCTGTAGCCAA-3′). The PCR reaction conditions were as follows: 1 cycle at 95 °C for 10 s, then 40 cycles at 95 °C for 5 s, at 60 °C for 30 s, at 95 °C for 1 min, at 60 °C for 30 s, and at 95 °C for 30 s. Relative mRNA levels of involucrin and CerS3 were normalized by GAPDH.
3
Quantitative RT-PCR for Viral RNA Detection
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The viral RNA copies in nasal or lung tissues of challenged mice were determined by Quantitative RT-PCR according to the protocol. Total RNA was extracted from 20 mg of lung tissues using a RNeasy Mini kit (Qiagen, Hilden, Germany, Cat.#74104). Then cDNA was synthesized using random primers and the SuperScript II RT kit (Invitrogen, Waltham, MA, USA, Cat.#18064014). Extracted RNA (10 µL) was reverse transcribed in a 20-µL reaction mixture containing 1 × first strand buffer, 100 mM DTT, 10 mM each dNTP, 50 ng of random primers, 40 U of RNaseOUT, and 200 U of SuperScript II RT at 42 °C for 50 min, followed by 15 min at 70 °C. The solution was incubated with RNase H (Invitrogen, Waltham, MA, USA, Cat.# 18021071) at 37 °C for 20 min. Synthesized cDNA was quantified using Power SYBR Green PCR Master Mix (Life Technologies, Carlsbad, California, United States, Cat.#4309155) in a 20-µL mixture containing 5 µL of cDNA (1/10), 10 µL of 2 × Power SYBR Green PCR Master Mix, 3 µL of RNase-free H2O, 10 µM forward primer and reverse primer in a Mx3000 QPCR System (Agilent, Santa Clara, California, USA).
4
Quantitative RT-PCR Analysis of DIP Genes
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Total RNAs were extracted from mid-exponential phase cultures following a single step RNA extraction procedure adapted from P. furiosus [40, 41] . The RNAs were treated with DNAse and further purified with the RNeasy kit from Qiagen according to the manufacturer's instructions. The absence of residual genomic DNA was verified by direct PCR amplification using gene targets listed in table 1. Total RNA were reverse transcribed using the RevertAid™ H Minus Reverse
Transcriptase kit (Fermentas, Lituany). The resulting cDNA was used as a template for target specific PCR amplification using primer pairs for each DIP genes (Table 1). RT-qPCR assays were performed on a Mx3000 QPCR system (Agilent Technologies, Santa Clara, CA, USA) using the Brilliant II Ultra-Fast SYBR ® Green QPCR master mix (Stratagene, La Jolla, CA, USA). The 20 µl reactions contained 1 µl of target cDNA (1 ng.µl -1 ), 0.3 µl of 1/500 diluted reference dye and 1 µmol.l -1 of each forward and reverse primers. Negative controls without template were included in each run. A specific cloned reference was used for each target gene. Transcripts levels were normalized to 16S rRNA genes. Values are reported as a ratio of expression levels relative to growth conditions under optimal temperature at atmospheric pressure.
Transcriptase kit (Fermentas, Lituany). The resulting cDNA was used as a template for target specific PCR amplification using primer pairs for each DIP genes (Table 1). RT-qPCR assays were performed on a Mx3000 QPCR system (Agilent Technologies, Santa Clara, CA, USA) using the Brilliant II Ultra-Fast SYBR ® Green QPCR master mix (Stratagene, La Jolla, CA, USA). The 20 µl reactions contained 1 µl of target cDNA (1 ng.µl -1 ), 0.3 µl of 1/500 diluted reference dye and 1 µmol.l -1 of each forward and reverse primers. Negative controls without template were included in each run. A specific cloned reference was used for each target gene. Transcripts levels were normalized to 16S rRNA genes. Values are reported as a ratio of expression levels relative to growth conditions under optimal temperature at atmospheric pressure.
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