Total RNA was isolated from kidney tissue stored at -80°C in TriPure solution. Total RNA concentrations were measured using a NanoDrop 1000 (Thermo Scientific, Waltham, MA, USA). The RNA was diluted to approximately 1000 µg/mL. cDNA was synthesized from each RNA sample using a thermal cycling machine and a reverse transcriptase cDNA synthesis kit (Roche Nano Lightcycler; Roche Diagnostics) according to the manufacturer's instructions. Epo, TNF-α, TNFR1, and TNFR2 mRNA levels were analyzed using β-actin as a reference gene. FAM-labeled primers/probes of the target genes and reference gene were amplified in a 20-μL reaction volume in 0.2-mL PCR tubes. Amplification was detected by using Dual Label TaqMan probes, and the measurements were performed using a Nano LightCycler (Roche Diagnostics). Final calculations were performed using the 2 -ΔΔct relative quantification method.
Lightcycler nano
Manufactured by Roche
Sourced in Germany, Switzerland, United States, Japan
The LightCycler Nano is a compact, real-time PCR instrument designed for fast, reliable, and sensitive nucleic acid quantification. It features a compact design and intuitive software interface for efficient sample analysis.
Automatically generated - may contain errors
Lab products found in correlation
Faststart essential dna green master, by Roche (25 mentions)
Rneasy mini kit, by Qiagen (20 mentions)
Trizol reagent, by Thermo Fisher Scientific (17 mentions)
Thunderbird sybr qpcr mix, by Toyobo (9 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (7 mentions)
Faststart essential dna green master mix, by Roche (7 mentions)
Trizol, by Thermo Fisher Scientific (7 mentions)
Quantitect reverse transcription kit, by Qiagen (6 mentions)
Revertra ace qpcr rt master mix, by Toyobo (5 mentions)
Rnaiso plus, by Takara Bio (5 mentions)
Primescript 2 high fidelity rt pcr kit, by Takara Bio (4 mentions)
Tri reagent, by Merck Group (4 mentions)
Itaq universal sybr green supermix, by Bio-Rad (4 mentions)
Light cycler 480 probe master system, by Roche (4 mentions)
Superscript 3 first strand synthesis system for rt pcr kit, by Thermo Fisher Scientific (4 mentions)
Rneasy midi kit, by Qiagen (4 mentions)
Revertra ace, by Toyobo (4 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (3 mentions)
Faststart essential dna green master kit, by Roche (3 mentions)
Rneasy kit, by Qiagen (3 mentions)
158 protocols using lightcycler nano
1
Quantifying Kidney Gene Expression
2
Quantification of Norovirus RNA by Real-Time RT-PCR
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Two-step TaqMan RT-PCR was performed with modifications (Kitajima et al., 2010 (link)). To synthesize cDNA, a High Capacity cDNA Reverse Transcription Kit (Life Technologies) was used. A 10-μl reaction mixture containing 5 μl RNA extract, 4 mM dNTPs, 1× RT Random Primers, 25 U of MultiScribe Reverse Transcriptase, and 10 U of RNase Inhibitor in 1× RT buffer was incubated at 25 °C for 10 min, at 37 °C for 120 min, and then at 85 °C for 5 min. TaqMan PCR master mixture was prepared in a 20 μl-reaction volume: 10 μl of 2× Premix Ex Taq (Takara Bio, Otsu, Shiga, Japan), 2 μl cDNA or plasmid standard, 2 μl of 4 μM each of the primers (forward, 5′-CCGCAGGAACGCTCAGCAG-3′; reverse, 5′-GGYTGAATGGGGACGGCCTG-3′), 0.5 μl of 12 μM TaqMan MGB probe (5′-FAM-ATGAGTGATGGCGCA-MGB/NFQ-3′, Life Technologies) and 5.5 μl of molecular grade water. PCR amplification was performed using a LightCycler Nano (Roche Applied Science) under the following conditions: initial denaturation at 95 °C for 30 s, followed by 40 cycles of amplification with denaturation at 95 °C for 15 s and annealing and extension at 60 °C for 60 s. The results were analyzed by software in LightCycler Nano. Quantification of viral cDNA in the reaction was calculated using a standard curve constructed by amplifying known amounts of MNV-S7-PP3 plasmid.
3
Quantitative Real-Time PCR Analysis
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Extraction of total RNA, RT-PCR analysis, and electrophoresis were carried out as described previously51 (link)52 (link). Quantitative real-time PCR was performed on a LightCycler Nano (Roche Diagnostics, Basel, Switzerland) using FastStart Essential DNA Green Master (Roche Diagnostics). Data analysis was performed using the LightCycler Nano software version 1.0 (Roche Diagnostics). mRNA expression levels were normalized against the level of Gapdh mRNA in the same sample. The evaluation of relative mRNA levels in each sample was carried out using the ΔΔCT method. Data are presented as fold changes relative to the control group. Primer sequences are listed in the Supplementary Table S4 .
4
Quantification of mRNA Levels in Adult Testes
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Reverse transcription quantitative PCR (RT-qPCR) analysis was performed to determine mRNA level of various target genes in adult testes. Total RNA was extracted from adult testes using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA synthesis from the total RNA was carried out using the PrimerScript II High Fidelity RT-PCR kit (Takara, Shiga, Japan) with random primers. RT-qPCR was performed using FastStart Essential DNA Green Master (Roche, Mannheim, Germany) and a LightCycler Nano (Roche, Basel, Switzerland). We used RP49 as a normalisation reference (Oka et al., 2015 (link)). Relative mRNA levels were quantified using LightCycler Nano software version 1.0 (Roche, Basel, Switzerland). The primer sets used were as follows: Rp49 forward, 5′-TTCCTGGTGCACAACGTG-3′; Rp49 reverse, 5′-TCTCCTTGCGCTTCTTGG-3′; Klp10A forward, 5′-GAATCTAGTCGTCTCGGCCAG-3′; Klp10A reverse, 5′-GCTTGTCGGACAGAAGATCGA-3′; Cp110 forward, 5′-CACGCCTCAACCATTTGTGAA-3′; Cp110 reverse, 5′-TCGAACTGCAGGATACGATCG-3′. Each sample was duplicated on the PCR plate, and the final results average three biological replicates. For quantification, the ΔΔCt method was used to determine the differences between the target gene expression relative to the reference Rp49 gene expression.
5
Quantifying GLA and G6PD mRNA Levels
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GLAp.R301Q/° fibroblasts were treated for 24 h. The cells were harvested and 2 µl of the crude RNA extract was reverse transcribed using the FastLane Cell cDNA kit (Qiagen, Hilden, Germany) according to the manufactureŕs specification. PCR samples were prepared with the FastStart Essential DNA Green Master kit (Roche, Mannheim, Germany) according to the manufacturer's specification. Primer sequences were 5′-TTCAAAAGCCCAATTATACAGAAA-3′ (forward) and 5′-CTGGTCCAGCAACATCAACA-3′ (reverse) for GLA and 5′-TGCCCCCGACCGTCTAC-3′ (forward) and 5′-ATGCGGTTCCAGCCTATCTG-3′ (reverse) for G6PD, respectively. PCR was carried out with the LightCycler® Nano (Roche, Mannheim, Germany) in combination with the LightCycler® Nano SW 1.1 software. Changes of mRNA amounts were calculated using the efficiency corrected relative quantification model [45 (link)].
6
Quantitative Gene Expression Analysis in Drosophila
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For quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) from the whole bodies, heads, and abdomens of adult flies fed diets with or without 2 mg/mL sesamin for 7 days. cDNA synthesis was performed using a PrimeScript II High Fidelity RT-PCR kit (Takara, Shiga, Japan) with oligo dT primers. qRT-PCR was performed using FastStart Essential DNA Green master mix (Roche, Mannheim, Germany) and a LightCycler Nano (Roche, Basel, Switzerland). RP49 was used as the normalization reference. Relative mRNA levels were quantified using the LightCycler Nano software version 1.0 (Roche, Basel, Switzerland). The primers used were as follows:
RP49-Fw, 5′-TTCCTGGTGCACAACGTG-3′,
RP49-Rv, 5′-TCTCCTTGCGCTTCTTGG-3′,
GFP-Fw, 5′-AAGCTGACCCTGAAGTTCATCTGC-3′,
GFP-Rv, 5′-CTTGTAGTTGCCGTCGTCCTTGAA-3′,
Nrf2-Fw, 5′-TTACATCTACGAGTACGCCGC-3′,
Nrf2-Rv, 5′-ACTGGAGCTCAAAACCGCTAA-3′,
Keap1-Fw, 5′-CCACCGTGGAGCGTTATGATA-3′,
Keap1-Rv, 5′-TTCCTGCATTCTGGACCAAGG-3′
7
Quantitative Gene Expression Analysis in Fly Models
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qRT-PCR analysis was performed to determine the expression level of lamin genes and Nups genes in adult flies with induced expression of each dsRNA with a bam-Gal4 driver as described (Yasukawa et al., 2015 (link)). Total RNA was extracted from adult flies using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA synthesis from the total RNA was carried out using a PrimeScript II High Fidelity RT-PCR kit (Takara, Shiga, Japan) with random primers. qRT-PCR was performed using FastStart Essential DNA Green Master (Roche, Mannheim, Germany) and a LightCycler Nano (Roche, Basel, Switzerland). RP49 was used as a normalization reference (Oka et al., 2015 (link)). Relative mRNA levels were quantified using LightCycler Nano software version 1.0 (Roche, Basel, Switzerland).
8
Bacterial Growth, RNA, and qPCR Analysis
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Bacterial growth, RNA purification, cDNA synthesis and quantitative PCR were performed as previously described (11 (link)). LightCycler Nano or LightCycler 96 systems (Roche) were used for qPCR runs. The data were analyzed with the LightCycler Nano or LightCycler 96 software packages (Roche) using the standard curve method. Expression values in the graphs are presented as relative to the mRNA levels of the reference gene gyrA.
9
Quantitative PCR Analysis of Testis Genes
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For qRT-PCR analysis, total RNA was extracted from young adult testes using the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). cDNA synthesis from the RNA was carried out using a PrimeScript II High Fidelity RT-PCR kit (Takara, Shiga, Japan) with oligo dT primers. qRT-PCR was performed using FastStart Essential DNA Green Master (Roche, Mannheim, Germany) and a LightCycler Nano (Roche, Basel, Switzerland). RP49 was used as a normalization reference [35 (link)]. Relative mRNA levels were quantified using LightCycler Nano software version 1.0 (Roche, Basel, Switzerland). The primers used were as follows: Nup62 (FW: 5′- TGAATTCGTTGCAGTGGATCG-3′, RV: 5′-TCTGGGAGTCTTGAATCTTGCC -3′), Nup58 (FW: 5′-TTCACGAATGTCAGCCACGA-3′, RV: 5′-ATCGCCTTGACGGTCTCTTG-3′), Nup54 (FW: 5′- TCTAGGTGTTGTGGAGGCTTTG-3′, RV: 5′-CGGGGTGGATTTTCAAGGTAC-3′), emb (FW: 5′-TCATTATGATCTCGCGCATGGC-3′, RV: 5′- TTCGCGCATGTTCTTGTACAGG-3′). Each sample was duplicated on the PCR plate, and the final results averaged three biological replicates. For the quantification, the ∆∆Ct method was used to determine the differences between target gene expression relative to the reference Rp49 gene expression.
10
Quantifying Histone Gene Expression in Flies
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For qRT-PCR analysis, total RNA was extracted from pharate adult flies using the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). cDNA synthesis from the RNA was carried out using a PrimeScript II High Fidelity RT-PCR kit (Takara, Shiga, Japan) with random primers. qRT-PCR was performed using FastStart Essential DNA Green Master (Roche, Mannheim, Germany) and a LightCycler Nano (Roche, Basel, Switzerland). RP49 was used as a normalization reference (Oka et al., 2015) . Relative mRNA levels were quantified using LightCycler Nano software version 1.0 (Roche, Basel, Switzerland). The primers used were as follows: histone H1 (L: 5'-AGTTGCAACGTCCGC TTC-3', R: 5'-TTGTGCCAGCAGATCCAG-3'), histone H2A (L: 5'-GAAGGGAAACTACGCAGAGC-3', R: 5'-AGCCAACTCG AGAACCTCAG-3'), histone H2B (L: 5'-TTCGTCGAAGGCGA TGAG-3', R: 5'-CGAGCGCTTGTTGTAGTGAG-3'), histone H3 (L: 5'-GAGCACCGAGCTTCTAATCC-3', R: 5'-CTTCCTG CAGAGCCATAACC-3'), histone H4 (L: 5'-GCGTCATCGCAA AGTACTGC-3', R: 5'-CCAGATATGCGCTTCACACC-3'). Each sample was duplicated on the PCR plate, and the final results average three biological replicates. For the quantification, the ∆∆Ct method was used to determine the differences between target gene expression relative to the reference Rp49 gene expression.
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