cDNA synthesis from RNA samples derived from embryonic myoblasts transduced with CMVGFP or CMVEMX2 was primed with oligo-dT and random hexamers. Amplification was performed in triplicate using qRT-PCR SYBR Green master mix including ROX passive reference dye (Applied Biosystems). Two reference genes, GAPDH and RPLO, were used for normalization of amplification variability across samples. Expression levels of each gene, calculated by determination of ΔΔCT, were compared between myoblasts expressing CMVGFP and CMVEMX2. Oligonucleotides used for amplification are listed in Supplement Table 1 .
Rox passive reference dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ROX™ Passive Reference Dye is a fluorescent dye used as a passive reference in real-time PCR (qPCR) experiments. It provides a stable fluorescent signal that is unaffected by the PCR reaction, allowing for normalization of the target signal.
Automatically generated - may contain errors
Lab products found in correlation
Quantstudio 6 flex, by Thermo Fisher Scientific (2 mentions)
Amplitaq gold dna polymerase, by Thermo Fisher Scientific (2 mentions)
Microamp enduraplate optical 96 well fast clear reaction plate with barcode, by Thermo Fisher Scientific (2 mentions)
Quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rna quantification kit, by Thermo Fisher Scientific (2 mentions)
Sybr green 1, by Thermo Fisher Scientific (2 mentions)
Steponeplus machine, by Thermo Fisher Scientific (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Amv rt, by Promega (1 mentions)
Dynamo colorflash sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
Quantstudio 12k flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
12 protocols using rox passive reference dye
1
Quantitative RT-PCR Analysis of Myoblast Transcripts
2
Mitochondrial DNA Damage Quantification
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Amplicons spanning 11kb of the 16.5kb mitochondrial DNA genome (Figure 1(A)) were amplified using a SYBR Green qPCR assay. This 11kb region spans 11 of the 13 genes encoding functional components of the electron transport chain (ETC) and a majority of the D-loop region which initiates mtDNA replication and transcription (Li et al., 2012) . SYBR Green I binds to double stranded mtDNA and fluoresces relative to a passive ROX reference dye. Fluorescence (ΔRn) increases exponentially to amount of amplicon, and is measured at the end of each cycle. As illustrated in Figure 1(B) data is analysed according the comparative Ct method (ΔΔCt) whereby a more damaged sample will require more cycles to produce the same amount of amplicon product (Santos et al., 2006) . The method was performed on a 96 well StepOnePlus™ machine (Applied Biosystems), with mastermix composition and settings outlined in Tables 2 and3. The Expand Long Range PCR System (Sigma) was used alongside SYBR Green I DNA dye (10,000X in DMSO; Lonza) and ROX passive reference dye (Applied Biosystems). To achieve 5X SYBR Green working concentration, a 1µl frozen aliquot was diluted in 2ml IDTE buffer. Each sample was assayed in triplicate with a standard deviation threshold of ≤ 0.4 Ct. Ct threshold was manually adjusted to the linear range on StepOnePlus™ v2.3 software, and melt curve analysis used to determine product size.
3
Microbiome 16S rDNA Amplification Protocol
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The microbial 16S rDNA was amplified through PCR in the following working solution: 10 µL of 2x Taqman Universal Master Mix II (AmpliTaq Gold DNA polymerase, deoxynucleotide triphosphates, ROX Passive Reference dye, optimized buffer components; ThermoFisher Scientific, Grand Island, NY), 2 µL (9 µM) forward primer, 2 µL (9 µM) reverse primer, 2 µL (2.5 µM) probe, 2 µL sample DNA, and 2 µL molecular grade water up to a final volume of 50 µL per reaction. The qPCR standard universal thermal cycling protocol was performed using the QuantStudio 6 Flex (ThermoFisher Scientific) as follows: activation of DNA polymerase at 50 °C for 2 minutes then increased to 95 °C for 10 minutes followed by 40 cycles of denaturation at 95 °C for 15 seconds followed by annealing/extension phase at 60 °C for 1 minute. Data collection occurred during the annealing/extension phase. Samples were plated in 384-well plates and covered with optical adhesive plastic. Plates were centrifuged at 2000 RPM for 2 minutes to remove bubbles from bottom of wells. All samples were run in triplicate with positive and negative controls.
4
Quantifying MHV68 Viral Loads via qPCR
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Viral genome loads were measured by quantitative real time PCR to detect ORF65 which encodes the capsid protein, M9 (Guo et al., 2009 (link)). DNA from murine blood, lungs, and spleen tissue (100 ng) was used to amplify MHV68 genomic coordinates 94,119 to 94,184 within ORF65 gene using StepOne Real-Time PCR System (Applied Biosystems). Primers ORF65F: 5′-GTC AGG GCC CAG TCC GTA-3′ and ORF65R: 5′-TGG CCC TCT ACC TTC TGT TGA-3′) overlapped a 65 bps long fragment. PCR mixture contained 0.5 μM of each primer and Maxima SYBR Green PCR reaction buffer with ROX-passive reference dye (Thermo Scientific) in the final volume of 20 μl. PCR program was as follows: 40 cycles of 94°C for 15 s, 58°C for 15 s, and 72°C for 15 s. qPCR standard curve was established using 10-fold serial dilutions of the MHV68 BAC DNA; 106 to 100 copies were used as templates in PCR mixtures that were amplified in parallel. Specificity of qPCR products was confirmed by melting curve analyses. PCR products were quantified by comparison with the standard curve. Viral genome equivalents in 1 ml of blood or 1 mg of tissue were determined from the mean of triplicate real-time PCR assays for each sample.
5
Quantitative RT-qPCR Analysis of Viral Transcripts
6
Thermal Shift Assay of PfCyRPA Protein
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DSF was performed in a Quant Studio 7 Flex Real-Time PCR System (Thermo Fisher Scientific), with excitation and emission wavelengths of 580 and 623 nm, respectively, using a MicroAmpTM EnduraPlateTM Optical 96-Well Fast Clear Reaction Plate with Barcode (Thermo Fisher Scientific). The samples were heated from 25°C to 90°C with stepwise increments of 0.016°C per second, followed by the fluorescence read-out. For each well, 20 µL final volume with 2 µg of PfCyRPA protein and 2-fold of ROX™ Passive Reference Dye (Thermo Fisher Scientific) was prepared with protein purification buffer. The assays were carried out in triplicates, and the results were analyzed in Protein Thermal ShiftTM Software V1.3.
7
Microbial 16S rRNA Gene Amplification
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The microbial 16S rRNA gene was amplified through a polymerase chain reaction (PCR) in the following working solution: 10μL of 2x Taqman Universal Master Mix II (AmpliTaq Gold DNA polymerase, deoxynucleotide triphosphates, ROX™ Passive Reference dye, optimized buffer components; ThermoFisher Scientific, Grand Island, NY), 2μL (9μM) forward primer, 2μL (9μM) reverse primer, 2μL (2.5μM) probe, 2μL sample DNA, and 2uL molecular grade water up to a final volume of 50μL per reaction.
V3_357F and V5_926R primers22 (link) modified with Nextera adaptors were developed in collaboration with the University of Minnesota Genomic Center in Minneapolis, MN.
V3_341 F_Nextera:
V5_926R_Nextera:
The RT-PCR standard universal thermal cycling protocol was performed using the QuantStudio™ 6 Flex (ThermoFisher Scientific) as follows: activation of DNA polymerase at 50°C for 2 minutes then increased to 95°C for 10 minutes followed by 40 cycles of denaturation at 95°C for 15 seconds followed by annealing/extension phase at 60°C for 1 minute. Data collection occurred during the annealing/extension phase. Samples were plated in 384 well plates and covered with optical adhesive. Plates centrifuged at 2000 RPM for 2 minutes to remove bubble from bottom of wells. All samples were run in triplicate with positive and negative controls.
V3_357F and V5_926R primers22 (link) modified with Nextera adaptors were developed in collaboration with the University of Minnesota Genomic Center in Minneapolis, MN.
V3_341 F_Nextera:
V5_926R_Nextera:
The RT-PCR standard universal thermal cycling protocol was performed using the QuantStudio™ 6 Flex (ThermoFisher Scientific) as follows: activation of DNA polymerase at 50°C for 2 minutes then increased to 95°C for 10 minutes followed by 40 cycles of denaturation at 95°C for 15 seconds followed by annealing/extension phase at 60°C for 1 minute. Data collection occurred during the annealing/extension phase. Samples were plated in 384 well plates and covered with optical adhesive. Plates centrifuged at 2000 RPM for 2 minutes to remove bubble from bottom of wells. All samples were run in triplicate with positive and negative controls.
8
Differential Scanning Fluorimetry of SARS-CoV-2 S Protein
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Differential scanning fluorimetry (DSF) was performed in a QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA), with excitation and emission wavelengths of 580 and 623 nm, respectively, using a MicroAmpTM EnduraPlateTM Optical 96-Well Fast Clear Reaction Plate with Barcode (Thermo Fisher Scientific, Waltham, MA, USA). The samples were heated from 25 °C to 90 °C with stepwise increments of 0.016 °C per second, followed by the fluorescence read out. For each well, 20 µL final volume with 4 µg of S protein and 4-fold of ROX™ Passive Reference Dye (Thermo Fisher Scientific, Waltham, MA, USA) was prepared with protein purification buffer. The assays were carried out in triplicates and the results were analyzed in the Protein Thermal ShiftTM Software V1.3.
9
Quantitative Real-Time PCR Optimization
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RT-qPCR was performed on a QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and ROX Passive Reference Dye (Affymetrix, Santa Clara, CA, USA). Amplifications were carried out under the following conditions: initial denaturation at 95 °C for 10 min followed by 40 cycles of 10 sec at 95 °C and for 1 min at the optimal annealing temperature. This was followed by a melting curve analysis in which the temperature raised from 65 °C to 95 °C in sequential steps of 0.05 °C for 1 s. Three technical replicates were performed for each biological sample, and the average cycle threshold (Ct) values of triplicates were calculated. Furthermore, no-template control was done in order to check whether primer-dimers or contamination with amplified PCR product were detectable. Five 5-fold serial dilution was made from cDNA samples to create a standard curve, and the amplification efficiency was determined for each candidate gene. The efficiency (E) values were calculated according to the equation: E = (10(−1∕slope) − 1) × 100, where slope is the slope of the standard curve (Radonić et al., 2004 (link)).
10
Laser Capture Microdissection for RNA Isolation
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Total RNA was isolated from the laser-captured hippocampal microvessels (300 microvessels per mice, n = 3 mice per diet/inhibitor group) utilizing an Arcturus PicoPure™ RNA Isolation Kit (Thermo Fisher Scientific, Santa Clara, CA, USA) in accordance to the manufacturer’s protocols. RNA quality of the LCM-derived microvessels was determined using Nanodrop. RNA quantification was conducted as per Affymetrix RNA quantification kit with SYBR Green I and ROX™ Passive Reference Dye protocol from Affymetrix, Santa Clara, CA. The entire amount of micro dissected tissue was subjected to RNA preparation for microarray analysis yielding 122.3 pico grams of RNA per array.
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