Liquid nitrogen-frozen muscle samples were homogenized, and RNA was extracted using TRIsure (Bioline) and the Quick-RNA Miniprep kit (Zymo Research) per manufacturer instructions. Cell populations were sorted directly into lysis buffer, and RNA was isolated using the Quick-RNA Microprep kit (Zymo Research). Complementary DNA (cDNA) was synthesized from 150 ng (sorted cells) or 1000 ng (whole muscle) of DNase-treated RNA using the SensiFAST cDNA synthesis kit (Bioline). Gene expression was quantified using TaqMan expression assay probes (Life Technologies) and 2x SensiFAST probe No-ROX mix (Bioline). All gene expression was normalized to 18 s unless otherwise noted.
Sensifast probe no rox mix
Manufactured by Meridian Bioscience
Sourced in Australia, United Kingdom
SensiFAST Probe No-ROX Mix is a 2x concentrated, ready-to-use master mix for probe-based real-time PCR. It contains all the necessary components for probe-based PCR, including a hot-start DNA polymerase, dNTPs, MgCl2, and stabilizers.
Automatically generated - may contain errors
Lab products found in correlation
Sensifast cdna synthesis kit, by Meridian Bioscience (3 mentions)
Qubit 3 fluorometer 3000, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Cfx connect system, by Bio-Rad (3 mentions)
Taqman expression assay probes, by Thermo Fisher Scientific (2 mentions)
Trisure, by Meridian Bioscience (2 mentions)
Quick rna miniprep kit, by Zymo Research (2 mentions)
Quick rna microprep kit, by Zymo Research (2 mentions)
Taqman array mouse immune fast 96 well plates, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Merck Group (1 mentions)
Smart cycler 2, by Cepheid (1 mentions)
Isolate 2 genomic dna kit, by Meridian Bioscience (1 mentions)
Dna extraction control 670, by Meridian Bioscience (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Cfx manager 3, by Bio-Rad (1 mentions)
Superscript 2 kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Deoxyribonuclease 1, by Thermo Fisher Scientific (1 mentions)
Cfx manager software version 3, by Bio-Rad (1 mentions)
Cfx96 touch deep well real time pcr detection system, by Bio-Rad (1 mentions)
10 protocols using sensifast probe no rox mix
1
RNA Extraction and Gene Expression Analysis
2
RNA Extraction and Gene Expression Analysis
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Liquid nitrogen-frozen muscle samples were homogenized, and RNA was extracted using TRIsure (Bioline) and the Quick-RNA Miniprep kit (Zymo Research) per manufacturer instructions. Cell populations were sorted directly into lysis buffer, and RNA was isolated using the Quick-RNA Microprep kit (Zymo Research). Complementary DNA (cDNA) was synthesized from 150 ng (sorted cells) or 1000 ng (whole muscle) of DNase-treated RNA using the SensiFAST cDNA synthesis kit (Bioline). Gene expression was quantified using TaqMan expression assay probes (Life Technologies) and 2x SensiFAST probe No-ROX mix (Bioline). All gene expression was normalized to 18 s unless otherwise noted.
3
Real-time PCR Assay for Acanella
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The real-time PCR used in this study was based on the TaqMan assay previously described by Qvarnstrom et al. [16 (link)]. The PCR primers and the TaqMan probe were purchased from Sigma-Aldrich. Reactions were prepared in a total reaction volume of 20 μL in smart cycler tubes (Cepheid) to contain 0.2 μM of forward primer AcanITSF1, 0.2 μM of reverse primer AcanITSR1, 0.05 μM of probe AcanITSP1, 2 μL of DNA template and 10 μL of SensiFast Probe No-ROX mix (Bioline). All reactions were prepared on a frozen block and upon completion of the reaction setup, were centrifuged for 10 seconds to move reaction contents to the bottom of the smart cycler tubes. All real-time PCR reactions were subjected to the following temperature cycling conditions: 40 cycles of 95°C for 15 seconds followed by 60°C for 45 seconds. Each PCR run was accompanied by negative controls consisting of DNA extracted from an uninfected mollusc and another that used 2 μL of H2O as template instead of DNA. All reactions were carried out on a Smart Cycler II (Cepheid).
4
Quantifying Colon Cancer NHE3 Expression
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TissueScan Colon Cancer cDNA Array III (Origene, Rockville, MD) was used to quantitate NHE3 gene expression by qPCR. The array plate was comprised of cDNA synthesized from RNA isolated from colonic biopsies from 24 matched pairs (48 samples) covering 24-normal, 5-Stage I, 5-IIA, 2-IIB, 2-IIIA, 3-IIIB, 2-IIIC, 1-III, 4-IV colorectal cancer cases. NHE3 and b-actin mRNA expression was analyzed using specific TaqMan primers/probe sets (Invitrogen) and Roche LightCycler96. In mice, expression of selected genes was analyzed by real-time qPCR. Total RNA (500 ng) purified from previously snap-frozen distal colon tissue (for details see “DNA/RNA Extraction ” section) was reverse transcribed with SensiFAST cDNA Synthesis Kit (Bioline) then 1 ml of the reaction was used for real-time qPCR with SensiFast Probe No-ROX Mix (Bioline), commercially available primers from Invitrogen, and Roche LightCycler96. All results were normalized to an endogenous reference GAPDH gene and to a calibrator calculated from Ct values from the control group.
5
DNA Extraction from Fluke Parasites
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Clean adult F. hepatica and F. gigantica flukes stored in 70% EtOH from the parasite collection at the Sydney School of Veterinary Science, University of Sydney were used as positive controls. Total genomic DNA from 1/5th of an adult fluke (25 mg, cut using a sterile scalpel blade) from each species was isolated using Isolate II Genomic DNA kit (BioLine, Australia) according to the manufacturer’s instructions and eluted in 100 μl of elution buffer (10 mM TrisCl buffer, pH = 8.5). To monitor DNA isolation efficiency and PCR inhibitors 5 μl of DNA Extraction Control 670 (Bioline, Australia) was included and DNA assayed for presence of extraction control signal on CFX96 Touch Real-Time PCR Detection System with the corresponding CFX Manager 3.1 software (BioRad, Australia) using SensiFAST Probe No-ROX Mix (BioLine, Australia) according to the manufacturer's instructions with expected CT values of <31. Each DNA isolation batch included a blank sample (ddH2O) to detect any potential contamination during the extraction process (extraction negative control). Extracted DNA was stored at -20°C prior to molecular analysis.
6
Quantification of Exosomal RNA by qPCR
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Total RNA was isolated from the extracted serum exosomes. First-strand complementary DNA (cDNA) was generated from normalized RNA using random hexamer primers and the Superscript II kit (Invitrogen). Primers were designed by the Roche Universal Probe Library Assay Design Center. Two units of sensiFAST probe No-ROX Mix (Bioline) were used in each 96-well reaction plate (Axon). A Roche LightCycler® 480 was used to perform the quantitative polymerase chain reaction (qPCR) and the real-time qPCR was repeated three times for each condition (25 (link)).
7
Immune Gene Expression in EPEC-Infected Mice
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The isolation of total RNA from colon tissues of EPEC-infected (presenting moderate or severe diarrhea) and control mice were performed by using a Qiagen RNeasy mini kit and QIAcube. cDNA was synthetized from 1µg of total RNA, quantified by Qubit 3 fluorometer 3000 (Invitrogen) and purified by deoxyribonuclease I (Invitrogen) treatment, with the iScript cDNA (Bio-Rad) as described by manufacturer instructions. qPCR was performed with 50 ng of cDNA in each well and SensiFAST probe no-ROX mix (Bioline) using a CFX Connect system (Bio-Rad) with the following conditions: 95°C for 2 min, 40 cycles of 95°C for 10 s and 60°C for 50 s. A pre-designed TaqMan array mouse immune fast 96-well plates (Applied Biosystems) was used to assess the expression of 92 genes listed in Supplementary Table 1
8
Quantitative Analysis of Immune Gene Expression in CDI-Infected Mouse Colon
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The isolation of total RNA from colon tissues of CDI-infected and control mice was performed by using a Qiagen RNeasy Mini Kit and QIAcube. cDNA was synthetized from 1 µg of total RNA, quantified by Qubit 3 Fluorometer 3000 (Invitrogen), and purified by deoxyribonuclease I (Invitrogen) treatment, with the iScript cDNA (Bio-Rad) as described by the manufacturer’s instructions. qPCR was performed with 50 ng of cDNA in each well and SensiFAST Probe No-ROX Mix (Bioline) using a CFX Connect system (Bio-Rad) with the following conditions: 95°C for 2 min, 40 cycles of 95°C for 10 s, and 60°C for 50 s. A predesigned TaqMan array mouse immune fast 96-well plate (Applied Biosystems) was used to assess the expression of all the genes shown in Figures 4A, B
9
Bacterial DNA Extraction and Antimicrobial Resistance Profiling
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Genomic DNA from bacterial isolates were extracted using the QIAGEN DNeasy Blood and Tissue Kit (Germantown, MD, USA). The genomic DNA concentration and purity were measured by Nanodrop 2000c Spectrophotometer (Thermo Scientific, MA, USA). The genomic DNA at a concentration between 1 and 10 ng/µl, was used as the DNA template for PCR and real-time PCR and was stored at − 20 °C until use.
Real-time PCR assays were performed as described previously to determine the presence of carbapenemase antimicrobial resistance genes (blaNDM and blaKPC) [22 (link)] and methicillin resistance gene (mecA) [23 (link)] using SensiFAST Probe No-ROX Mix (Bioline, London, UK) on CFX96 Touch Deep Well™ Real-Time PCR Detection System (Bio-Rad, CA, USA). Analysis was performed using Bio-Rad CFX Manager software version 3.1.
PCR assays were performed as described previously to determine the presence of ESBL genes [24 (link)], carbapenemase genes (blaOXA-23,24,51,58) [25 (link)], colistin resistance genes (mcr-1 to mcr-9) [26 , 27 ], vancomycin resistance genes [28 ], and Enterococcus speciation (ddlE) [28 ] using the AmpliTaq® Gold DNA polymerase (Thermo Fisher Scientific, USA) on Mastercycler® nexus (Eppendorf, Inc, USA).
Real-time PCR assays were performed as described previously to determine the presence of carbapenemase antimicrobial resistance genes (blaNDM and blaKPC) [22 (link)] and methicillin resistance gene (mecA) [23 (link)] using SensiFAST Probe No-ROX Mix (Bioline, London, UK) on CFX96 Touch Deep Well™ Real-Time PCR Detection System (Bio-Rad, CA, USA). Analysis was performed using Bio-Rad CFX Manager software version 3.1.
PCR assays were performed as described previously to determine the presence of ESBL genes [24 (link)], carbapenemase genes (blaOXA-23,24,51,58) [25 (link)], colistin resistance genes (mcr-1 to mcr-9) [26 , 27 ], vancomycin resistance genes [28 ], and Enterococcus speciation (ddlE) [28 ] using the AmpliTaq® Gold DNA polymerase (Thermo Fisher Scientific, USA) on Mastercycler® nexus (Eppendorf, Inc, USA).
10
Immune Response Profiling in EPEC-Infected Mice
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The isolation of total RNA from colon tissues of EPEC-infected (presenting moderate or severe diarrhea) and control mice were performed by using a Qiagen RNeasy mini kit and QIAcube. cDNA was synthetized from 1µg of total RNA, quantified by Qubit 3 fluorometer 3000 (Invitrogen) and purified by deoxyribonuclease I (Invitrogen) treatment, with the iScript cDNA (Bio-Rad) as described by manufacturer instructions. qPCR was performed with 50 ng of cDNA in each well and SensiFAST probe no-ROX mix (Bioline) using a CFX Connect system (Bio-Rad) with the following conditions: 95 °C for 2 min, 40 cycles of 95 °C for 10 s and 60°C for 50 s. A pre-designed TaqMan array mouse immune fast 96-well plates (Applied Biosystems) was used to assess the expression of 92 genes listed in supplementary Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. All fold changes were determined using the ΔΔC t method [81] (link).
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