RNA was purified using TRI reagent (Thermo Scientific) according the to manufacturer’s protocol. RNA samples were treated with DNase I (Thermo Scientific) according to the manufacturer’s protocol. First-strand cDNA synthesis was carried out using the Maxima First Strand cDNA synthesis Kit for qPCR (Thermo Scientific) according to the manufacturer’s protocol. qPCR reactions were prepared using gene-specific primers (Supplementary file 2d ) and Platinum SYBR Green qPCR Supermix-UDG (Thermo Scientific) according to the manufacturer’s protocol. An AriaMx Real-time PCR System (Agilent Technologies) was used for quantification of RNA levels and the X0 method was used for calculations of relative RNA levels (Thomsen et al., 2010 (link)) normalized to either GAPDH or beta-actin (ACTB) mRNA as indicated.
Maxima first strand cdna synthesis kit for qpcr
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Maxima First Strand cDNA Synthesis Kit for qPCR is a reagent kit used for the reverse transcription of RNA into complementary DNA (cDNA) for downstream quantitative PCR (qPCR) applications. The kit contains all the necessary components, including reverse transcriptase enzyme, reaction buffer, and random hexamer primers, to efficiently generate first-strand cDNA from total RNA or mRNA samples.
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Lab products found in correlation
Dnase 1 treatment, by Thermo Fisher Scientific (3 mentions)
Tri reagent, by Thermo Fisher Scientific (2 mentions)
Dsdnase, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Platinum sybr green qpcr supermix udg, by Thermo Fisher Scientific (1 mentions)
Ariamx real time pcr system, by Agilent Technologies (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Rneasy lipid tissue kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Spectrum plant total rna kit, by Merck Group (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
Biotaq dna polymerase, by Meridian Bioscience (1 mentions)
Ibrightfl1000 system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Cfx384 thermocycler, by Bio-Rad (1 mentions)
Gelred nucleic acid gel stain, by Biotium (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
11 protocols using maxima first strand cdna synthesis kit for qpcr
1
Gene Expression Quantification Protocol
2
RNA Isolation and cDNA Synthesis Protocol
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The RNA isolation and cDNA synthesis procedure were performed as described previously49 (link). In brief, total RNA was isolated from mouse cells or cell lines with TRIzol Reagent (Ambion, 15596-018) and subjected to cDNA synthesis using Maxima First Strand cDNA synthesis Kit for qPCR (Thermo Scientific, K1642). RNA from purified human adipocytes was extracted using the RNeasy Lipid Tissue kit (Qiagen, 74804), and subjected to cDNA synthesis using the High Capacity RNA-to-cDNA kit (Applied Biosystems, 4387406). mRNA relative amounts were measured using the CFX96 Real-Time System (Bio-Rad Laboratories Inc.) and calculated by normalization to the level of cyclophilin mRNA (mouse cells or cell lines), or to the level of GAPDH mRNA (human cells). The primer sequences that were used for quantitative real-time PCR analyses are provided in Supplementary Table 3 .
3
Tissue Sampling and RNA Extraction for Wound Healing
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Tissues were harvested 0, 4, 6, 8, 10, 14, and 28 days post wounding from three trees to provide three biological replicates, following the method explained above, and ground in liquid nitrogen. Total RNA was extracted from each powdered wound-healing tissue using the SpectrumTM Plant Total RNA Kit (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions and stored at −80 °C until use. The RNA was DNase treated (Turbo DNase, Invitrogen, Waltham, MA, USA) following the manufacturer’s instructions. RNA purity was assessed using 1% agarose gel electrophoresis and OD260/280 and OD260/230 ratios using a NanoDrop ND-1000 UV-VIS spectrophotometer (Thermo Fisher Scientific, Inc., Ottawa, ON, Canada). Subsequent synthesis of cDNA was carried out using Maxima® First Strand cDNA Synthesis kit for qPCR (Thermo Fisher Scientific Inc., Ottawa, ON, Canada).
4
Quantitative and Qualitative RNA Analysis
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Total RNA was extracted using TRIzol (Life Technologies). Reverse transcription was performed using random priming and the Maxima First Strand cDNA Synthesis Kit for qPCR, with dsDNase (Thermo Fisher Scientific), according to the manufacturer’s guidelines. qPCR was performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific), 10 mM of each primer and 20 ng of cDNA, in a CFX384 thermocycler (Bio-Rad). Variations in RNA input were corrected by analyzing the expression of GAPDH (human), β-actin (mice), or rp49 (Drosophila) as a housekeeping gene. The ΔΔCt method was used for relative quantification. Primers sequences are presented in Supplemental Table 5 .
Splicing assays were performed by reverse-transcription PCR (RT-PCR), using BIOTAQ DNA Polymerase (Bioline). The primers used have been described previously and were selected to give a length difference of 10%–25% between exon-inclusion and exon-exclusion products (62 (link)). PCR amplification was performed for 20–24 cycles, and PCR products were resolved on agarose gels, stained with GelRed Nucleic Acid Gel Stain (Biotium), visualized using an iBright FL1000 system (Invitrogen), and quantified using ImageJ (NIH).
Splicing assays were performed by reverse-transcription PCR (RT-PCR), using BIOTAQ DNA Polymerase (Bioline). The primers used have been described previously and were selected to give a length difference of 10%–25% between exon-inclusion and exon-exclusion products (62 (link)). PCR amplification was performed for 20–24 cycles, and PCR products were resolved on agarose gels, stained with GelRed Nucleic Acid Gel Stain (Biotium), visualized using an iBright FL1000 system (Invitrogen), and quantified using ImageJ (NIH).
5
Bacterial RNA Extraction and RT-qPCR
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Bacterial RNA was extracted from overnight cultures using the ISOLATE II RNA Mini Kit (Bioline Reagents Ltd, London, UK) according to the manufacturer’s protocol. DNase I treatment (Ambion by life technologies, Carlsbad, USA) was performed both on-column and on the final eluted RNA. The concentration of the RNA was determined using Nanodrop and the integrity of the RNA was verified on a 1% agarose gel. One μg of RNA was reverse transcribed using Maxima First Strand cDNA Synthesis Kit for qPCR (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s protocol. A minus-RT control reaction was set up for each sample to rule out residual DNA and at least two independent RNA preparations were used. For real-time qPCR, 1/2000 of the cDNA synthesis reaction was used.
6
Bacterial RNA Extraction and qPCR Analysis
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The bacterial RNA was extracted using the ISOLATE II RNA Mini kit (Bioline Reagents Ltd, London, UK) according to the manufacturer’ s protocol, with two DNase I treatments (Ambion by life technologies, Carlsbad, USA): on column and on the final RNA preparation. The concentration of RNA was measured using NanoDrop (NanoDrop ND-1000, Thermo Fisher Scientific). The integrity of RNA was tested by running 0.5 µg RNA on a 1% agarose gel with 3% chlorine. For cDNA synthesis, 500 ng of RNA was reverse transcribed using the Maxima First Strand cDNA Synthesis Kit for qPCR (Thermo Fisher Scientific). For the qPCR to determine expression of virulence genes, 1 µl of 10× diluted cDNA was used. Further experiments were performed on four biological extraction replicates.
7
Quantifying Hippocampal Gene Expression
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Total RNA extraction of hippocampus samples was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA concentrations of the samples were firstly quantified using the NanoDropTM spectrophotometer 24 (Thermo Fisher, Waltham, MA), followed by the reverse transcription and qualitative polymerase chain reaction. 1 μg of RNA was reverse transcribed to cDNA using the Maxima First Strand cDNA Synthesis Kit for q-PCR (Thermo Scientific) on the PCR machine (BioRad, Hercules, CA). qPCR was then performed using GoTaq qPCR Master Mix (Promega, Madison, WI) on the ABI 7500 Real-Time PCR system (Applied Biosystems, Waltham, MA). 10 ng of cDNA, 0.2 μM primers and GoTaq qPCR Master Mix was used for each reaction and three replicates were performed for each gene. To analyze the data, the threshold cycles depicting the gene expression levels were normalized to GAPDH, a housekeeping gene (ΔCt). This is then followed by the normalization to the control group (ΔΔCt), to achieve the final fold change in gene expression. Primers used were: GAPDH forward primer: 5′-CATCACTGCCACCCAGAAGACTG-3′, GAPDH reverse primer: 5′- ATGCCAGTGAGCTTCCCGTTCAG-3′; IL-1b forward primer: 5′-CACAGCAGCACATCAACAAG-3′, IL-1b reverse primer: 5′-GTGCTCATGTCCTCATCCTG-3′.
8
Bacterial RNA Extraction and cDNA Synthesis
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Bacterial RNA was extracted from overnight cultures using the ISOLATE II RNA Mini Kit (Bioline Reagents Ltd., London, UK) according to the manufacturer’s protocol. DNase I treatment (Ambion by life technologies, Carlsbad, CA, USA) was performed both on-column and on the final eluted RNA. The concentration of RNA was determined using Nanodrop, and RNA integrity was verified on a 1% agarose gel. A Maxima First Strand cDNA Synthesis Kit for qPCR (Thermo Fisher Scientific, Waltham, MA, USA) was used to reverse transcribe 1 µg of RNA according to the manufacturer’s protocol. A minus-RT control reaction was set up for each sample to rule out residual DNA. For qPCR 1/2000 of the cDNA synthesis reaction was used. At least two independent RNA preparations were used for real-time qPCR.
9
Bacterial RNA Extraction and Reverse Transcription
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Bacteria from overnight cultures were first treated with 4 μg/ml lysozyme in TE buffer for 1 h at 37°. Bacterial RNA was thereafter prepared using TRIreagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. The RNA was DNase treated twice using dsDNase (Thermo Fisher Scientific) with phenol/chisam extraction and ethanol precipitation performed in between. The concentration of the RNA was determined using Nanodrop, and the integrity of the RNA was verified on a 1% agarose gel. A total of 100 ng of RNA was reverse transcribed (RT) using Maxima First Strand cDNA Synthesis Kit for qPCR (Thermo Fisher Scientific) according to the manufacturer’s protocol. A control reaction without RT was set up for each sample to rule out residual DNA. The cDNA synthesis reaction was diluted in water and 1/2000 was used for qPCR. At least three independent RNA preparations were used for qPCR.
10
Viral Detection in Animal Tissues
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For all animal tissue samples (Figs. 1 –3 and Extended Data Figs. 1a,b,d and 2 ) RNA was isolated as mentioned earlier and cDNA was prepared using the Maxima First Strand cDNA Synthesis Kit for qPCR (catalogue no. K1672; Thermo Fisher Scientific). The primers used for EDIM, MNV-1, murine astrovirus, WU23, MNV-3, MNV-4 and CR6 are listed in Supplementary Table 2 . cDNA obtained from the samples was run along with the corresponding primers and with SYBR Green (catalogue no. 1725124; Bio-Rad Laboratories) in the Roche LightCycler 96 System (catalogue no. 05815916001). The thermal cycling conditions included a pre-incubation step 95 °C for 90 s followed by 45 cycles at 95 °C for 10 s, 54 °C for 10 s and 72 °C for 110 s.Each sample were run in duplicate for each experiment. For EDIM, MNV-1, WU23, MNV-3, MNV-4 and CR6, standards were run along with each experiment to measure virus genome copies per milligram of tissue. A detailed description of the standard curve preparation for each virus is given in the section ‘Standard curve preparation for EDIM, MNV-1, CR6, WU23, MNV-3, MNV-4’.
For murine astrovirus (Extended Data Fig.1d ), samples were analysed with the Gapdh housekeeping gene (Supplementary Table 2 ) along with a mock-infected sample. The fold change obtained was measured over the mock-normalized by cycle threshold (Ct) values obtained for the housekeeping gene.
For murine astrovirus (Extended Data Fig.
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