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11 protocols using taqman universal master mix

1

mRNA Quantification by RT-qPCR

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After total RNA was extracted and mRNA purified, mRNA was converted to cDNA using the TransScriptor First-Strand cDNA Synthesis SuperMix (TransScript, #AT301, Beijing, China). The assays-on-demand primers and probes and TaqMan Universal Master Mix were used to examine gene expression by the Roche LC480 Sequence Detection System (TransStart) according to the user’s manual. The house-keeping gene ACTB (encoding β-actin) was used as internal control. All reactions were carried out in triplicate and relative expression levels were calculated as 2−△△CT after normalization to the internal control. Each sample was analyzed independently three times.
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2

Multiplex PCR for Mycobacterium paratuberculosis

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Multiplex PCR targeting the insertion element IS900 of the Map genome was performed. The total reaction volume was 20 µL containing 5 µL of DNA template, 1× TaqMan Universal MasterMix (Roche Diagnostics, USA), 0.2 µM of each primer (Roche Diagnostics), and 0.1 µM of FAM probe (Roche Diagnostics). The sequences of the two primers designed to amplify a 63-nucleotide fragment of the IS900 target gene were gacgcgatgatcgaggag (F) and gggcatgctcaggatgat (R). The reactions were processed in a LightCycler 2.0 system (Roche Diagnostics) under the following standard conditions: one cycle at 95℃ for 10 min, 45 cycles at 95℃ for 10 sec, 60℃ for 30 sec, and 72℃ for 1 sec; and cooling at 40℃ for 30 sec. Negative and positive controls (Mycobacterium avium subsp. paratuberculosis ATCC 19698) were included. A positive DNA extraction control was also included. The system performed a presence-absence assay for which only the measurements of the last cycle were recorded. Real-time PCR curves of normalized fluorescence for FAM exceeding a threshold value of 0.01 at less than 40 cycles were considered positive (cycle to positive [CP]) as long as the curves had a normal and expected shape.
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3

Quantification of sncRNA715 in Mice Sciatic Nerve

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Sciatic nerves were prepared from P 1, 4 or 9 C57BL/6J mice as described above, dissociated in 700μl Qiazol using a Tissue Ruptor (Qiagen) and total RNA was extracted using the miRNeasy Mini Kit (Qiagen). Reverse transcription of mRNAs was performed with the Transcriptor High Fidelity Reverse Transcription Kit and qPCR was performed with the Taqman Universal Master Mix (all Roche Applied Science). SncRNA715 was reverse transcribed by the TaqMan MicroRNA Reverse Transcription Kit with stem-loop RT primers specific for sncRNA715 or snoRNA135 sequence (Applied Biosystems, order no. sncRNA715 PN4427975, snoRNA135 PN440887) and amplified with the Taqman Universal Master Mix (Roche Applied Sience) with specific primers and probes for the indicated sncRNAs (Applied Biosystems). The crossing points were used for relative quantification based on the ΔΔCt method using REST software [27 ]. SnoRNA135 was used as a reference gene. PCR products and the 10 bp DNA Step Ladder (Promega) were separated on 4% agarose gels and stained with ethidium bromide.
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4

Gene Expression Analysis by RT-PCR

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Real-time Polymerase Chain Reaction (PCR) was performed with a LightCycler 480-II Instrument (Roche Diagnostics, Risch-Rotkreuz, Switzerland) with TAQMAN Universal Master Mix (Roche). Gene expression was calculated relative to the housekeeping gene (RPL13A). Sequence primers, probes, and PCR conditions are shown in Supplementary Table 1.
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5

Transcriptional Profiling of Oligodendrocytes

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Total RNA of Oli-neu cells and primary OPCs were extracted using either the RNeasy Mini Kit or miRNeasy Mini Kit (both Qiagen). Reverse transcription of mRNAs was performed with the Transcriptor High Fidelity Reverse Transcription Kit (Roche Applied Science). SncRNA715 was reverse transcribed by the TaqMan MicroRNA Reverse Transcription Kit with stem-loop RT primers specific for sncRNA715 sequence (Applied Biosystems) and amplified with the Taqman Universal Master Mix (Roche Applied Science) with specific primers and probes for sncRNA715 (Applied Biosystems). RT-PCR primers specific for rat MBP: 5′-AACATTGTGACACCTCGAACA-3′ and 5′-TGTCTCTTCCTCCCCAGCTA-3′. PCR of Ago1-4 mRNAs was carried out using the Pfu-DNA Polymerase according to manufacturer’s protocol. Primers were specifically designed to differentiate the four different murine Ago proteins (Ago1: 5′-AGCGGCAGGTGCCTAC-3′ and 5′-GGATCTGGCCACTGACC3-′, Ago2: 5′-ATATGCC TTCAAACCTCCACC-3′ and 5′-CCAGGGCCTGGATCGTC-3′, Ago3: 5′-ACAAGCCTGTCAGCACTAAC-3′ and 5′-CAGAC TTCTAGTGGCAGGTATG-3′, Ago4: 5′-GGAGCTCCTCTACA GCCAGG-3′ and 5′-CAAGCCGGGCATAATATGC-3′. Ago1-4 PCR products and 100 bp DNA Step Ladder (Promega) were separated on 1% agarose gels and stained with ethidium bromide (EtBr). RT-PCR products of Mbp mRNA and sncRNA715 were separated on 4% agarose gels and stained with EtBr.
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6

Quantitative PCR of Ca. Clavichlamydia salmonicola

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Quantification of Ca. Clavichlamydia salmonicola was done using qPCR via standard curve of 10 X dilution series, following protocol and primers from Mitchell et al. (2010 (link)). Briefly, the ClavoRTf, ClavoRTfr primers and ClavoRTp Taqman® probe (5′‐Fam‐CGAACTCCAGATGCCTGGAGCCG‐BHQ‐3′) (Thermo Fisher, USA) were used in a 20 μl real‐time PCR reaction containing: 1 μl of each primer (10 μM), 0.5 μl of probe (10 μM), 10 μl of TaqMan Universal Master mix (Roche) and 2 μl of DNA template (~10 ng μl). qPCR was carried out in triplicate in a real‐time PCR machine (System Quant Studio 5™, USA) under the following conditions: 50°C for 2 min, 95°C for 10 min followed by 50 cycles of 95°C for 30 s and 60°C for 1 min.
The detection limit for Ca. Clavichlamydia salmonicola was 1000 copies due to increased variation between replicates in the standard curve for low DNA concentration (Ct SD >0.5) (Forootan et al., 2017 (link)). Prevalence at time point was determined by the percentage of positive samples per time point and infection intensity reflects the number of copies based on qPCR. Standard curve for quantification of Ca. Clavichlamydia salmonicola had a slope −2.974, ɣ‐intercept 39.7 and efficiency 116.8.
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7

RNA Extraction and RT-PCR Analysis

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The RNA extraction was achieved using TRIzol (Life Technologies) according to the manufacturers protocol. 0.5 µg of RNA was reversely transcribed into cDNA using an NZY First-Strand cDNA Synthesis Kit (NZYTech, Lisbon, Portugal) following the manufacturer´s instructions. The RT-PCR was performed in a LightCycler 480-II Instrument (Roche, Mannheim, Germany) using TaqMan Universal Master Mix (Roche). Analysis of the results was carried out using Qbase+ version 2.5 software (Biogazelle, Ghent, Belgium). Gene expression was calculated relative to the housekeeping gene ribosomal protein L13A (RPL13A). Sequence primers, probe, and PCR conditions are available upon request.
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8

Cardiac Tissue RNA Extraction and Gene Expression Analysis

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A 30 mg sample of frozen cardiac tissue was collected and RNA was extracted using TRIzol reagent (Invitrogen), in accordance with the manufacturer's protocol. Using 2 μg aliquots of RNA, cDNAs were synthesized using the NZY First-Strand cDNA Synthesis kit (NZYTech, Lisbon, Portugal), following the manufacturer's instructions.
The PCR was performed in a LightCycler 480-II Instrument (Roche, Basel, Switzerland) using TaqMan Universal Master Mix and specific Universal ProbeLibrary (UPL) probes (Roche) of each gene (MPC1, MPC2). PCR was performed in a 10 μl reaction mixture volume with 20 μM forward primer and 20 μM reverse primer for mRNA. The PCR cycling was as follows: a denaturing step of 10 min at 95°C, followed by 50 amplification cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s, followed by cooling to 40°C. Analysis of the results was carried out using Qbase + version 2.5 software and the relative expression levels of genes were estimated by the 2−ΔΔCT method (30 (link)) as the average value of each sample normalized to the geometric average of the housekeeping genes GAPDH, HPRT1, and ACTB, selected after doing a geNorm study. DNA primer sequences and UPL probes are described in Table 1.
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9

RT-PCR Analysis of Gene Expression

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RNA extraction was achieved using TRIzol® (Sigma-Aldrich), following the manufacturer’s protocol. A total of 0.5 mg of RNA was reverse transcribed into cDNA using Super Script VILO (Thermofisher Scientific, Waltham, MA, USA), following the manufacturer´s instructions. RT-PCR was developed in a LightCycler 480-II Instrument (Roche, Mannheim, Germany) using TaqMan Universal Master Mix (Roche). The analysis of the results was carried out using Qbase+ version 2.5 software (Biogazelle, Ghent, Belgium). Gene expression was calculated relative to the housekeeping gene Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). Sequence primers (Table 3) and PCR conditions were described; a pre-incubation step at 95 °C for 5 min, followed by 40 cycles corresponding to amplification (denaturation at 95 °C for 10 seconds (s), annealing all the primers at 60 °C for 10 s with an extension at 72 °C for 1 s), and the last step was cooling at 4 °C for 5 min. Only a single acquisition mode was applied during the extension step in the amplification process.
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10

Leptospira DNA Detection via qPCR

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Tissue samples were subject to a DNA extraction–purification protocol using the High Pure PCR Template Preparation kit (Roche, Indianapolis, IN, USA), following the manufacturer’s instructions.
The DNA templates obtained from the above protocol were analyzed in a qPCR system (Roche LightCycler 2.0) using a TaqMan probe targeting the LipL32 gene [22 (link)], which is specific only for pathogenic Leptospira spp. The primers lipL32-45F (5′-AAG CAT TAC CGC TTG TGG TG-3′) and lipL32-286R (5′-GAA CTC CCA TTT CAG CGA TT-3′) were used to amplify a fragment of 242 bp, which was detected by the above-mentioned probe, LipL32-189P (FAM-5′-AA AGC CAG GAC AAG CGCCG-3′-BHQ1). The amplification mixture for each sample contained 0.7 μM primers, 0.15 μM probe, 10 μL Master Mix TaqMan universal (Roche), and 5 μL DNA template in a total volume of 20 μL. Samples were amplified with the following program: (1) initial denaturation at 95 °C for 2 min, (2) 40 cycles of denaturation for 5 s at 95 °C, and (3) annealing/elongation for 30 s at 58 °C. The system contained a negative and positive control in order to survey the proficiency of the reaction in addition to negative and positive DNA controls.
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