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Lightcycler nano instrument

Manufactured by Roche
Sourced in United States, Switzerland, Germany

The LightCycler Nano Instrument is a real-time PCR system designed for fast, precise, and efficient nucleic acid amplification and quantification. It features a compact and user-friendly design, supporting a wide range of sample types and applications.

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46 protocols using lightcycler nano instrument

1

Isolation and qPCR Analysis of Inflammatory Genes

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Mice were euthanized 15 h after the i.a. injection of MSU, the knee joint samples were collected, homogenized in 500 μl of Trizol reagent and centrifuged (12,000 rcf, 15 min, 4°C). Total RNA was extracted using the SV Total RNA Isolation System (Promega) (Verri et al., 2008 (link)) and measured with a spectrophotometer and the wavelength absorption ratio (260/280 nm) was between 1.8 and 2.0 for all preparations. qPCR was performed in a LightCycler Nano Instrument (Roche, Mississauga, ON, United States) sequence detection system using the Platinum SYBR Green qPCR SuperMix UDG (Invitrogen, United States). The mRNA level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primers used were Gapdh forward: CAT ACC AGG AAA TGA GCT TG, reverse: ATG ACA TCA AGA AGG TGG TG; gp91phox (NADPH oxidase sub-unity), forward: AGC TAT GAG GTG GTG ATG TTA GTG G, reverse: CAC AAT ATT TGT ACC AGA CAG ACT TGA G; nlrp3, forward: AGC TAT GAG GTG GTG ATG TTA GTG G, reverse: CAC AAT ATT TGT ACC AGA CAG ACT TGA G; pro-il-1β, forward: GAA ATG CCA CCT TTT GAC AGT G, reverse: TGG ATG CTC TCA TCA GGA CAG; cyclooxygenase-2 (COX-2), forward: GTG GAA AAA CCT CGT CCA GA, reverse: GCT CGG CTT CCA GTA TTG AG. The SYBR green PCR Master Mix was used according to the manufacturer’s instructions.
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2

Quantitative Analysis of Stress Genes

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Using TRIzol reagent (Invitrogen, Waltham, MA, USA), total RNA was extracted from the 3rd-instar larvae of each genotype. cDNA was synthesized from total RNA using an oligo dT primer and a PrimeScriptTM High Fidelity RT-PCR Kit (TaKaRa, Clontech Laboratories, Shiga, Japan). Real-time PCR was carried out using FastStart Essential DNA Green Master Mix and a Light Cycler Nano Instrument (both from Roche, Mannheim, Germany). The qPCR primers were synthesized as follows: RP49-Fw, 5′-TTCCTGGTGCACAACGTG-3′ and RP49-Rv, 5′-TCTCCTTGCGCTTCTTGG-3′; TotA-Fw, 5′-CCCAGTTTGACCCCTGAG-3′ and TotA-Rv, 5′-GCCCTTCACACCTGGAGA-3′; TotB-Fw, 5′-CGCATGGCTCCTAGCTTAAGA-3′ and TotB-Rv, 5′-CTGGGTACTCCATCGACCATG-3′; TotC-Fw, 5′-TACTATGCCTTGCCCTGCTCC-3′ and TotC-Rv, 5′-TGTTCAGGGGACAACGTGGG-3′; TotF-Fw, 5′-AGGCACGTCAAATGCTCGC-3′ and TotF-Rv, 5′-TGTTGGTTGTTGTGTGCCCG-3′. Each sample was triplicated, and the final PCR results were obtained after averaging three biological replicates. The ∆∆Ct method was used to determine the differences between the expression of target genes relative to that of the reference gene, Rp49. Real-time PCR was performed on a LightCyclerNano using the FastStart Essential DNA Green master kit (Roche)
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3

Quantitative Gene Expression Profiling

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The total RNA was extracted from third instar larvae with each genotype while using the Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). cDNA synthesis from the total RNA was carried out using the PrimeScriptTM High Fidelity RT-PCR Kit (TaKaRa, Clontech Laboratories, Shiga, Japan) using an oligo dT primer. Real-time PCR was performed while using the FastStart Essential DNA Green Master (Roche, Mannheim, Germany) and a Light Cycler Nano instrument (Roche). According to software the Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus.cgi), the primers for qRT-PCR were synthesized as follows: RP49-Fw,5′-TTCCTGGTGCACAACGTG-3′; RP49-Rv,5′-TCTCCTTGCGCTTCTTGG3′; histoneH1-FW,5′-AGTTGCAACGTCCGCTTC-3′; histoneH1-RV,5′-TTGTGCCAGCAGATCCAG-3′; histoneH2A-Fw,5′-GAAGGGAAACTACGCAGAGC-3′; histoneH2A-Rv,5′-AGCCAACTCGAGAACCTCAG-3′; histoneH2B-Fw, 5′-TTCGTCGAAGGCGATGAG-3′; histoneH2B-Rv 5′-CGAGCGCTTGTTGTAGTGAG-3′, histoneH3-Fw, 5′-GAGCACCGAGCTTCTAATCC-3′; histoneH3-Rv 5′- CTTCCTGCAGAGCCATAACC-3′, histoneH4-Fw, 5′-GCGTCATCGCAAAGTACTGC-3′; histoneH4-Rv; 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 expressions relative to the reference Rp49 gene expression.
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4

Quantification of RXRα Expression in Umbilical Cord Samples

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Five micrograms of total RNA from umbilical cord samples were synthesized to cDNA using the Transcriptor First-Strand cDNA synthesis kit (Roche) following the manufacturer’s instructions. Assay efficiency was calculated using a dynamic range of cDNA dilution series (1:1, 1:2, 1:10, 1:100, 1:1,000, and 1:10,000). Quantitative real-time PCR was performed using the LightCycler TaqMan Master Kit (Roche), and TaqMan gene expression assay probes for RXRα and β-actin were purchased from Thermo Fisher Scientific. qPCR conditions for RXRα gene expression were the following: for amplification, 10 min at 95°C followed by 45 cycles of 10 s at 95°C, 30 s at the primer annealing temperature (60°C), and 10 s at 72°C. qPCR assays were performed three times in duplicate using a LightCycler Nano Instrument (Roche). Gene expression data were normalized using β-actin expression. Fold change in expression was calculated using the 2−ΔΔCt method.
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5

Quantification of Immune Gene Expression

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Total RNA was extracted from the 3rd instar larvae of each genotype using the Trizol® reagent (Invitrogen). Synthesis of cDNA from the total RNA was carried out using the PrimeScriptTM High Fidelity RT-PCR Kit (Takara Bio) and oligo dT primers. RT-PCR was performed using the FastStart Essential DNA Green Master (Roche) and a Light Cycler Nano instrument (Roche). The qRT-PCR primers were synthesised as follows: RP49-Fw, 5′-TTCCTGGTGCACAACGTG-3′; RP49-Rv, 5′-TCTCCTTGCGCTTCTTGG-3′; Drosomycin-Fw, 5′-GTACTTGTTCGCCCTCTTCG-3′; Drosomycin-Rv, 5′-CAGGGACCCTTGTATCTTCC-3′; Defensin-Fw, 5′-CTTCGTTCTCGTGGCTATCG-3′; Defensin-Rv, 5′-CCAGGACATGATCCTCTGGA-3′; Diptericin-Fw, 5′-CAGTCCAGGGTCACCAGAAG-3′; Diptericin-Rv, 5′-AGGTGCTTCCCACTTTCCAG-3′; Metchnikowin-Fw, 5′-TACATCAGTGCTGGCAGAGC-3′; Metchnikowin-Rv, 5′-ACCCGGTCTTGGTTGGTTAG-3′; AttacinA-Fw, 5′-ACTACCTTGGATCTCACGGGA-3′; AttacinA-Rv, 5′-TGATGAGATAGACCCAGGCCA-3′; Cecropin-Fw, 5′-ATCGGAAGCTGGTTGGCTAAA-3′; Cecropin-Rv, 5′-CTGGGTACTCCATCGACCATG-3′. Each sample was duplicated on the PCR plate and the final results average three biological replicates. For the quantification, the ΔΔCt method was used to determine the difference between target gene expression relative to the reference Rp49 gene expression.
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6

Rapid Leptospira Detection via qPCR

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Real-time PCR was carried out by targeting the 241 bp region of the lipL32 gene, which encodes the major outer membrane of pathogenic Leptospira spp. [12 (link),13 (link),14 (link)]. The master mix (20 µL) was prepared from Fast Start DNA probe master (Roche, Switzerland), primer and probes (LipL32-45F, LipL32-286R, Probe-189), and 5 µL of DNA primer. Amplification was done by the LightCycler® nano instrument (Roche Diagnostics International Ltd., Rotkreuz, Switzerland) at 45 cycles. Positive samples of the lipL32 gene were molecularly and genetically identified.
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7

Transcriptional response of Bradyrhizobium to herbicides

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After Bradyrhizobium sp. RD5-C2 was precultivated in HM medium to reach the stationary phase, it was exposed to 100‍ ‍μM 2,4-D (1 day) and 100‍ ‍μM 2,4,5-T (1 and 3 days) with shaking. Total RNA was extracted using ISOGEN-LS (Nippon Gene) according to the manufacturer’s instructions. RNA samples were then treated with DNase I (Takara Bio), and 1‍ ‍μg of each treated sample was used for cDNA synthesis using the PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio). Real-time PCR was performed using FastStart Essential DNA Green Master (Roche Diagnostics) and LightCycler Nano Instrument (Roche Diagnostics) according to the manufacturer’s instructions. The sig gene for sigma factor was used as an internal control. Primer sets were C2cad1A227-f/C2cad1A436-r (cadA1, 210‍ ‍bp), C2cad2A548-f/C2cad2A779-r (cadA2, 232‍ ‍bp), C2tfdAa308-f/C2tfdAa488-r (tfdAa, 181‍ ‍bp), and C2sig1288-f/C2sig1454-r (sig, 167‍ ‍bp). The PCR reaction was as follows: 95°C for 10‍ ‍min followed by 45 cycles at 95°C for 10‍ ‍s, 57°C for 10‍ ‍s, and 72°C for 15 s. The expression level of each gene was normalized to that of the sig (sigma factor) gene using the 2–ΔCT (ΔCT=Ct target gene–CT sig) calculation for statistical analyses (Dunnett’s test [P<0.05]). Three biological experiments were conducted for each treatment, and three real-time PCR reactions were performed for each experiment.
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8

Quantifying Gene Expression in Hepatic Cells

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RNA from unstretched and stretched hepatic ECs, human hepatocytes and HepG2 cells was isolated using the High Pure RNA Isolation Kit (Roche, 11828665001) and cDNA was synthesized using the Super-ScriptTM II Reverse Transcriptase (Thermo Fisher Scientific, 18064014) according to the protocols provided by the companies. The following primer sequences (purchased from Eurogentec) were used: Human MYDGF forward 5′–TCG TGC ATT CCT TCT CCC AT–3′ and human MYDGF reverse 5′–ACC TCT GCC TTG AAC TGT GT–3′; human AFP forward: 5´–ACA TCC TCA GCT TGC TGT CT–3′ and human AFP reverse 5´–AAT GCT TGG CTC TCC TGG AT–3′; human RPLP0 forward 5′–GAA GAC AGG GCG ACC TGG AA–3′ and human RPLP0 reverse 5′–CCA CAT TGT CTG CTC CCA CA–3′; Human B2M forward 5′–TTT CAT CCA TCC GAC ATT GA–3′ and human B2M reverse 5′–CCT CCA TGA TGC TGC TTA CA–3′; Human HPRT forward 5′–GCA GAC TTT GCT TTC CTT GG–3′ and human HPRT reverse 5′–AAC ACT TCG TGG GGT CCT TT–3′. Quantitative real-time PCR was run in duplicates by using the LightCycler Nano instrument (Roche) with the LightCycler® Nano Software 1.1 (Roche) or the QuantStudioTM 1 Real-Time PCR Instrument (Applied Biosystems, A40425) with the QuantStudioTM Design & Analysis Software v1.5.1 (Thermo Fisher Scientific). Relative gene expression of MYDGF or AFP was normalized to housekeeping genes and calculated by using the comparative CT method76 (link).
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9

Validating RNA Sequencing with qRT-PCR

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To validate RNA sequencing results, quantitative reverse transcription-PCR (qRT-PCR) was performed on selected genes using a LightCycler Nano Instrument (Roche, Basel, Switzerland) with the FastStart Essential DNA Green Master (Roche) and specific primers for sigA (sigAf/sigAr), nosR (nosRf/nosRr), nosZ (nosZf/nosZr), napA (napAf/napAr), nirK (nirKf/nirKr), norB (norBf/norBr), narK (narKf/narKr), nasC (nasCf/nasCr), and bll3385 (bll3385f/bll3385r) (Table S2). The PCR program was set according to the manufacturer’s instructions. The specificity of PCR amplification was confirmed by a melting-curve analysis. This analysis was performed in duplicate for each of the two independent RNA samples. Expression levels calculated by the 2−ΔΔCt method (33 (link)) were normalized to the sigA level and expressed relative to the values for the wild type.
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10

Hippocampal Gene Expression Analysis

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The mRNA expression of genes encoding brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and N-methyl-d-aspartate (NMDA1) in the hippocampus were determined by real-time PCR. Total RNA was isolated from the hippocampus using a column-based RNeasy Mini Kit (QIAGEN, Hilden, Germany) and was reverse-transcribed into cDNA using reverse transcriptase (M-MLV, Thermo Fisher Scientific) with oligo(dT)12–18 primer. The quantitative real-time PCR using SYBR Green was conducted to determine the relative expression levels for the respective genes, using LightCycler® Multiplex Masters (Roche, Basel, Switzerland) with the appropriate primer sets (Table 2) on a LightCycler® Nano Instrument (Roche). The transcript expression levels were normalized to the expression level of the β-actin [19 (link)].
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