For quantitative RT-PCR, total RNA was isolated from mouse lungs 7 d.p.i. and reverse transcribed into cDNA as described [47 (link),49 (link)]. Primers to specific murine genes (described in Supporting Information Table S2 ) were used to amplify transcript cDNA and relative transcript copies determined by the ΔΔCT method with an actin internal control by SyberGreen (Applied Biosystems, Carlsbad, CA, USA) real-time detection on a LightCycler thermocycler (Roche, Indianapolis, IN, USA). Significance of relative gene expression was determined by an unpaired, 2-tailed t-test. Viral DNA representing viral genome copy number was determined for each mouse BAL sample by qPCR with primers specific to MHV-68 genomic ORF65/M9 or ORF57 loci as previously described [48 (link),51 (link)].
Lightcycler thermocycler
Manufactured by Roche
Sourced in France, Germany, United States
The LightCycler thermocycler is a laboratory instrument designed for real-time polymerase chain reaction (PCR) analysis. It provides precise temperature control and rapid cycling capabilities for the amplification and detection of DNA or RNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Lightcycler software version 4, by Roche (5 mentions)
Pcr lightcycler dna master sybrgreen reaction mix, by Roche (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
M mlv reverse transcriptase, by Promega (3 mentions)
Sybr ex taq, by Takara Bio (2 mentions)
Miniprep plus r2072, by Zymo Research (2 mentions)
Tri reagent, by Zymo Research (2 mentions)
Lightcycler version 4, by Roche (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Sybergreen, by Thermo Fisher Scientific (1 mentions)
Magna pure compact kit, by Roche (1 mentions)
Liaison system, by DiaSorin (1 mentions)
Genotype helicodr, by Hain Lifescience (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Rq1 dnase, by Promega (1 mentions)
Oligo dt primer, by Thermo Fisher Scientific (1 mentions)
Faststart universal sybr green master mix, by Roche (1 mentions)
26 protocols using lightcycler thermocycler
1
Quantitative RT-PCR Analysis of Murine Lung Gene Expression
2
Measles Case Reporting Protocol
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We reported the main biological parameters. Lymphocytes and platelet counts were expressed in billions per liter (G/L); normal values were between 1.24–3.56 and 150–445 G/L, respectively. Sodium and potassium were expressed in mmol/L; normal values were 135–145 and 3.5–5 mmol/L, respectively. C-reactive protein (CRP) was expressed in mg/L and was considered increased if >5 mg/L.
We reported liver function through the values of alanine aminotransferase (ALT; normal value <55 UI/L) and aspartate aminotransferase (AST; normal value <34 UI/L). We reported acute renal dysfunction (creatinine >90 μmol/L in patients with prior normal renal function). Oxygen partial pressure (pO2) was expressed in millimeters of mercury (mmHg), and hypoxemia was defined as a pO2 <80 mmHg.
Measles serology was performed by chemiluminescence immunoassay (CLIA) analysis with the LIAISON system (DiaSorin, Saluggia, Italy) for IgM and IgG, with qualitative and semiquantitative detection (IgG threshold value of 15 UA/mL). Nucleic acids were extracted using the MagNA Pure Compact Kit (Roche, Penzberg, Upper Bavaria, Germany). RNA amplification was performed with a LightCycler thermocycler (Roche).
We reported liver function through the values of alanine aminotransferase (ALT; normal value <55 UI/L) and aspartate aminotransferase (AST; normal value <34 UI/L). We reported acute renal dysfunction (creatinine >90 μmol/L in patients with prior normal renal function). Oxygen partial pressure (pO2) was expressed in millimeters of mercury (mmHg), and hypoxemia was defined as a pO2 <80 mmHg.
Measles serology was performed by chemiluminescence immunoassay (CLIA) analysis with the LIAISON system (DiaSorin, Saluggia, Italy) for IgM and IgG, with qualitative and semiquantitative detection (IgG threshold value of 15 UA/mL). Nucleic acids were extracted using the MagNA Pure Compact Kit (Roche, Penzberg, Upper Bavaria, Germany). RNA amplification was performed with a LightCycler thermocycler (Roche).
3
Molecular detection of H. pylori resistance
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Fluoroquinolone and clarithromycin resistance were tested by using GenoType® HelicoDR(Hain Life Science, Germany) test kit as described by the following the manufacturer's description. The point mutations related to the clarithromycin resistance was detected by using a real-time PCR- (RT-PCR-) based hybridization assay. H. pylori 23SrRNA gene-specific primers with probes shown in Table 1 were used, and melting curve analysis was done. For the RT-PCR experiments and hybridization reactions, LightCycler thermocycler (Roche Diagnostics, Neuilly Sur Seine, France) was used.
Real-time PCR reactions were done by using glass capillary tubes with 20 μl volume. 3 μl template DNA, 0.4 μl forward and reverse primers (20 μM each), 0.2 μl sensor and anchor probes (20 μM each), and 2 μl of FastStart DNA Master Hybridization Probes (Roche Diagnostics) were included into each tube. The RT-PCR reaction conditions were 95°C for 10 min as an initial denaturation, 95°C for 0 s for 50 amplification cycles, 60°C for 10 s as annealing, and 72°C for 17 s as an extension. Also, a melting curve step was added to the reaction composed of 95°C for 0 s, 45°C for 30 s to cool (temperature transition rate of 20°C/s), and in the final step, a slow increase in the temperature to 85°C at a rate of 0.1°C/s with the continuous acquisition of fluorescence decrease [11 (link)].
Real-time PCR reactions were done by using glass capillary tubes with 20 μl volume. 3 μl template DNA, 0.4 μl forward and reverse primers (20 μM each), 0.2 μl sensor and anchor probes (20 μM each), and 2 μl of FastStart DNA Master Hybridization Probes (Roche Diagnostics) were included into each tube. The RT-PCR reaction conditions were 95°C for 10 min as an initial denaturation, 95°C for 0 s for 50 amplification cycles, 60°C for 10 s as annealing, and 72°C for 17 s as an extension. Also, a melting curve step was added to the reaction composed of 95°C for 0 s, 45°C for 30 s to cool (temperature transition rate of 20°C/s), and in the final step, a slow increase in the temperature to 85°C at a rate of 0.1°C/s with the continuous acquisition of fluorescence decrease [11 (link)].
4
Helicobacter pylori Detection and Clarithromycin Resistance Screening
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An in-house qPCR assay was used to detect both the presence of H. pylori and point mutations conferring clarithromycin resistance as described [12 (link)]. From the homogenized suspension, DNA was isolated by using a QIAamp DNA mini kit (Qiagen SA, Courtaboeuf, France). A 267-bp fragment of the H. pylori 23S rRNA gene was amplified with primers for HPYS and HPYA (Table 1 ) using the LightCycler thermocycler (Roche Diagnostics, Neuilly sur Seine, France). The specificity of the qPCR method was evaluated by using different microorganisms (Enterococcus faecalis, Pseudomonas aeruginosa, coagulase-negative Staphylococcus, Candida albicans, Escherichia coli, Klebsiella pneumoniae) for which qPCR testing remained negative. The qPCR testing was also negative for Campylobacter spp. Each run included positive and negative controls, the former prepared from 10−2, 10−4, and 10−6 dilutions of 45 μg/mL DNA from H. pylori strain H37Rv, and the latter consisting of sterile water. Quality control was acceptable when the negative control had an undetectable cycle threshold (Ct) and the 10−2, 10−4, and 10−6 dilutions of H. pylori DNA McFarland 1 had Ct values of 17–19, 19–27, and 27–33, respectively [13 (link)].
5
Quantifying Gene Expression in Tissues
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Total RNA from cells and murine and human tissues was isolated using direct-zol RNA MiniPrep Plus R2072 from ZymoResearch according to the manufacturer’s instructions. Mice tissue and intestinal resections were homogenated with TRI Reagent® (ZymoResearch, Irvine, CA, USA), using the GentleMACS Dissociator (Milteny Biotech, Gladbach, Germany) as previously described [36 (link)]. cDNA was obtained from previously isolated RNA by reverse transcription PCRusing the the PrimeScript RT reagent Kit (Takara Bitechnology, Dalian, China). Gene expression was analyzed by real-time Quantitative PCR using SYBR® Ex Taq (Takara Bio Inc., Saint-Germain-en-Laye, France) in LightCycler thermocycler (Roche Diagnostics, Mannheim, Germany). Specific oligonucleotides detailed in Table 3 and Table 4 were designed to perform the analysis. The relative gene expression, as fold increase, was expressed as follows: change in expression (fold) = 2 − Δ(ΔCT) where ΔCT = CT (target) − CT (housekeeping) and Δ(ΔCT) = ΔCT (treated) − ΔCT (control), where β-actin was the housekeeping gene used.
6
Quantifying mRNA Levels in Adipose Tissue
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Total RNA was isolated from adipose tissue using Trizol reagent (Invitrogen, Carlsbad, CA) and quantified by absorbance at 260 nm. One microgram total RNA was reverse transcribed with 200U M-MLV reverse transcriptase (Promega, Madison, WI) and 300 pmol oligo-dT. Real-time PCR was performed in 20 mL final volume containing 50 ng cDNA, PCR LightCycler-DNA Master SYBRGreen reaction mix (Roche, Indianapolis, IN), 4 mM MgCl2, and 0.5mM each specific primer using a LightCycler thermocycler (Roche, Indianapolis, IN). Controls without reverse transcription were included to ensure that amplification was from mRNA and not from genomic DNA. Amplicons were characterized according to their melting temperature, determined by LightCycler thermocycler, and after their size had been evaluated after being electrophoresed on agarose gel. Each target gene’s mRNA level was standardized against the GAPDH mRNA level for each sample. The Ct method was used for mRNA quantification, expressed as arbitrary units (a.u.).
7
Quantification of Stem Cell Markers by Q-PCR
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Primers for the ALDH1 region (forward 5′-GAGTGTTGAGCGGGCTAA-3′ and reverse 5′-CTCCTCCACATTCCAGTTTG-3′), CD44 region (forward 5′-GTCGAAGAAGGTGTGGG-3′ and reverse 5′-GGTCTGGAGTTTCTGACG-3′), CD47 region (forward 5′-CTCCTTCGTCATTGCCATA-3′ and reverse 5′-AACTAGTCCAAGTAATTGTGCT-3′), and β-glucuronidase GUS region (forward 5′-CTCCTTCGTCATTGCCATA-3′ and reverse 5′-CCTTTAGTGTTCCCTGCTAGAATA-3′) were used for gene quantification. All oligo primers were synthesized by Genomics BioSci and Tech (Taipei, Taiwan). A LightCycler thermocycler (Roche Molecular Biochemicals, Mannheim, Germany) was used for Q-PCR analysis. One microliter of sample and master mix (Roche, Basel, Switzerland) was first denatured for 10 min at 95 °C and then subjected to 40 cycles (denaturation at 95 °C for 5 s; annealing at 60 °C for 5 s; and elongation at 72 °C for 10 s) with detection of fluorescence intensity. Gene expression was finally normalized to GUS expression using the built-in Roche LightCycler Software, version 4 [21 (link)].
8
Quantifying PPM1F Expression in Breast Tumors
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Total RNA from human breast tumors and normal tissue samples acquired directly from patients was isolated with TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol [21 (link)]. The PPM1F-specific PCR primers were synthesized as Forward: 5′-GCCTACTTTGCTGTGTTTGA-3′ and Reverse: 5′-TCTCGCTTGGCTTTCCT-3′, and the β-glucuronidase-specific primers were synthesized as Forward: 5′-AGTGTTCCCTGCTAGAATAGATG-3′ and Reverse: 5′-AAACAGCCCGTTTACTTGAG-3′. A LightCycler thermocycler (Roche Molecular Biochemicals, Mannheim, Germany) was used for quantitative real-time PCR. The PPM1F mRNA fluorescence intensity was measured and normalized to glucuronidase expression through the built-in software (Roche LightCycler Version 4) [21 (link)].
9
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from 100 mg of tissue using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and quantified according to its absorbance at 260nm (NanoDrop 2000, Thermo Scientific, Wilmington, USA). Contaminating genomic DNA was degraded with DNAse RQ1 (Promega, Madison, USA), cDNA was synthesized from one microgram of total RNA using 300 pmol oligo-dT primers, 10 mM dNTP (Thermo Scientific, Wilmington, USA) and 200 U M-MLV reverse transcriptase (Promega, Madison, USA). Real-time PCR was performed in a final volume of 20 uL containing 50 ng cDNA, 3mM MgCl2, PCR LightCycler-DNA Master SYBRGreen reaction mix (Roche, Indianapolis, USA) and 0.5 mM of each specific primers (Table 1 ). Amplification was performed in a using LightCycler thermocycler (Roche, Indianapolis, USA). Controls without reverse transcription were included to ensure that amplifications were from mRNA and not from genomic DNA. Amplicons were characterized according to their melting temperature and size. The mRNA level of each target gene was calculated using the 2ΔΔCt method and normalized against the mRNA of β-actin.
10
Quantifying Lentiviral Transduction Efficiency
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Primers for the WPRE (Woodchuck Hepatitis Virus Response Element) region (forward 5′-TCATGCTATTGCTTCCCGTA-3′ and reverse 5′-CCAAGGAAAGGACGATGAT-3′) were used for lentivirus quantification. All oligo primers were synthesized by Genomics BioSci and Tech (Taipei, Taiwan). A LightCycler thermocycler (Roche Molecular Biochemicals, Mannheim, Germany) was used for qPCR analysis. One microliter of sample and master mix (Roche FastStart Universal SYBR Green Master Mix, Roche Applied Science, Mannheim, Germany) was first denatured for 10 min at 95 °C and then subjected to 40 cycles (denaturation at 95 °C for 5 s; annealing at 60 °C for 5 s; and elongation at 72 °C for 10 s) with detection of fluorescence intensity. All the PCR samples underwent a melting curve analysis to detect non-specific PCR products. Luciferase gene expression from the qPCR analysis was normalized to GUS (β-glucuronidase) expression as an indicator of DNA input using the built-in Roche LightCycler Software, version 4 (Roche Molecular Biochemicals, Mannheim, German).
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