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10 protocols using sensifast sybr no rox qpcr kit

1

Transcriptional Analysis of Fluconazole Resistance

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RNA was extracted from cultures growing exponentially in 23.43μM fluconazole using the QIAGEN RNeasy® kit. 1μg of isolate was treated with DNAse and analyzed using an Agilent Bioanalyzer to quantify nucleic acid concentration and verify purity. cDNA synthesis was performed using a combination of oligo-DT and random hexamer primers using the Thermo Scientific™ Maxima™ H Minus First Strand cDNA Synthesis Kit. qPCR on these samples was then performed using a Bioline SensiFAST™ SYBR No-ROX qPCR kit and Ct values were quantified using a CFX machine. cDNA synthesis and qPCR was performed for PDR5 and UBC6 (which acted as loading control).
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2

Transcriptional Analysis of Fluconazole Resistance

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RNA was extracted from cultures growing exponentially in 23.43μM fluconazole using the QIAGEN RNeasy® kit. 1μg of isolate was treated with DNAse and analyzed using an Agilent Bioanalyzer to quantify nucleic acid concentration and verify purity. cDNA synthesis was performed using a combination of oligo-DT and random hexamer primers using the Thermo Scientific™ Maxima™ H Minus First Strand cDNA Synthesis Kit. qPCR on these samples was then performed using a Bioline SensiFAST™ SYBR No-ROX qPCR kit and Ct values were quantified using a CFX machine. cDNA synthesis and qPCR was performed for PDR5 and UBC6 (which acted as loading control).
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3

qPCR Analysis of Zebrafish Gene Expression

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Prior to qPCR analysis, RNA was treated with DNase I (Thermo Fisher Scientific, NH, USA) to remove possible traces of genomic DNA according to the manufacturer's instructions. After DNase treatment, RNA was reverse transcribed into cDNA with a Reverse Transcription kit (Fluidigm, CA, USA) according to the manufacturer's instructions. Gene expression was measured by using SsoFast EvaGreen Supermix with Low ROX qPCR kit (Bio-Rad, CA, USA) with the CFX96 qPCR system (Bio-Rad, CA, USA). Zebrafish genes were normalized with expressed repetitive element loopern4 (Vanhauwaert et al., 2014 (link)) and compared with the average induction of pooled baseline sample of healthy non-infected zebrafish. Results were analyzed using the ΔCt method and are shown as fold induction.
Mycobacterial loads were measured with the SensiFAST SYBR No-ROX qPCR kit (Bioline, London, UK) from genomic DNA according to the manufacturer's instructions. Each bacterial quantification qPCR run included standard curve of known amounts of M. marinum DNA. Primer sequences and gene association numbers are shown in Table 1.
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4

qRT-PCR Analysis of Luciferase Reporters

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For qRT-PCR analysis, cells were lysed with cytoplasmic RNA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM KCl, 0.5% Triton X-100, 0.2 mM Pefabloc), clarified and split for luciferase and qRT-PCR assay. RNA was isolated with TriFast FL (Peqlab), treated with RQ1 DNase I, and reverse-transcribed using the Maxima first strand cDNA synthesis kit (Thermo Fisher). RLuc and FLuc were quantified using previously described primers (15 (link)) and sensiFAST SYBR No ROX qPCR kit (Bioline). Relative RLuc/FLuc expression levels were calculated using ΔΔCt method.
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5

Quantifying Mycobacterial celA1 Expression

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Prior to qPCR analysis, RNA was reverse transcribed into cDNA with a reverse transcription kit (Fluidigm, CA, USA) according to the manufacturer’s instructions. celA1 expression was measured using soFast EvaGreen supermix with the low ROX qPCR kit (Bio-Rad, CA, USA) and the CFX96 qPCR system (Bio-Rad). The primers used for celA1 were (forward) 5′-ACACTCCGCAGTCCTACT-3′ and (reverse) 5′-TAGAGCGTCAGAATCGGC-3′. The number of mycobacterial cells in the sample was quantified using the SensiFAST SYBR no-ROX qPCR kit (Bioline, London, UK) on bacterial DNA according to the manufacturer’s instructions. The primers used for Mmr quantification were targeted against the 16S-23S locus AB548718 (forward, 5′-CACCACGAGAAACACTCCAA-3′; reverse, 5′-CACCACGAGAAACACTCCAA-3′). Each bacterial quantification qPCR run included a standard curve of the known amounts of Mmr DNA. The mycobacterial cell number in each sample was used to normalize celA1 expression.
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6

Quantifying Neuronal Gene Expression

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For RT–qPCR analysis, RNA was isolated with TRIzol (Thermo Fisher Scientific), treated with RQ1 DNase I and reverse-transcribed using the Maxima first-strand cDNA synthesis kit (Thermo Fisher Scientific). Ago2, Hbs1l and Gapdh were quantified using sensiFAST SYBR No ROX qPCR kit (Bioline). Homer and Snord15b were used to estimate the efficiency of soma/neurite separation. See Supplementary Table 7 for primer sequences. Relative expression levels were calculated using the ΔΔCt method with Gapdh as a reference gene.
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7

Quantifying mRNA expression in neurons

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RNA from soma and neurites was treated with RQ1 DNase I to remove traces of gDNA and reverse-transcribed using the Maxima first strand cDNA synthesis kit (Thermo Fisher). qPCR was performed with sensiFAST SYBR No ROX qPCR kit (Bioline) with the primers provided in Supplementary Table S1. RT-qPCR reactions were carried out in technical duplicates and biological triplicates (soma) or duplicates (neurites), using a CFX96 Real-Time PCR system (Biorad). Relative neurites/soma expression levels were calculated using ΔΔCt method, with Capdh as a reference RNA.
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8

Quantitative RT-PCR Analysis of Neurite Transcripts

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RNA from soma and neurites was treated with RQ1 DNase I, reverse-transcribed using the Maxima first strand complementary DNA synthesis kit (Thermo Fisher), and quantified by quantitative PCR (qPCR) using sensiFAST SYBR No ROX qPCR kit (Bioline). The following primers were used (PrimerBank ID): Nxf7 (13561071a1), Vangl1 (29164511a1), ldlrap1 (160333774c1), Col3a1 (20380522a1), Crtap (225543172c1), Tagln (291045204c1), Bmper (24371215c1), Lamb1 (114326496c1), Myo1c (124494243c3), Mme (31543255a1), Stx3 (924268a1), Nid2 (26343027a1), Mbnl2 (140971799c1), St3gal6 (118130739c1), Mov10 (254540178c1), H3f3a (6680159a1), H2afy2 (133892300c1), Gng3 (6754022a1), Fbll1 (148539881c1), Nup210 (172073151c1), Tubb3 (12963615a1), rRNA (fw: 5′-aaacggctaccacatccaag-3′, rev: 5′-cctccaatggatcctcgtta-3′). Relative neurites/soma expression levels were calculated using ΔΔCt method, with rRNA as a reference RNA.
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9

Quantitative RT-qPCR Analysis of Gene Expression

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Total RNA from the early and late passages were extracted with a Zymo Quick-RNA MiniPrep kit (R1054, Zymo Research, Tustin, CA, USA) according to the manufacturer’s instructions. The RNA concentration and purity were evaluated with a NanoDrop One spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The SensiFAST cDNA synthesis kit (BIO-65053, SensiFAST, Bioline, Memphis, TN, USA) was used to synthesize cDNA from RNA. Each PCR reaction was conducted in a 20 μL reaction mixture with a final amount of 10 ng cDNA for each mixture using the SensiFAST SYBR No-ROX qPCR kit (BIO-98005, Bioline, Memphis, TN, USA). Quantitative RT-qPCR experiments were carried out with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Berkeley, CA, USA). The cycling parameters were as follows: 95 °C for 2 min, followed by 40 cycles at 95 °C for 5 s, an optimized annealing temperature for 10 s, and 72 °C for 5 s. The results of each target gene were normalized to β-actin mRNA expressions. The primer sets were designed using the Primer3 online software (https://primer3.ut.ee/) (accessed on 21 November 2021). The list of primers used for the experiments is shown in Table S1. Each experiment had three replicates per condition, and a statistical analysis was performed with a Mann–Whitney t-test using GraphPad Prism 9.3.1.
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10

RT-qPCR Analysis of Neuronal Transcripts

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For RT-qPCR analysis, RNA was isolated with Trizol (Thermo Fisher), treated with RQ1 DNase I, and reverse-transcribed using the Maxima first strand cDNA synthesis kit (Thermo Fisher). Ago2 and Gapdh were quantified using sensiFAST SYBR No ROX qPCR kit (Bioline). Homer and Snord15b were used to estimate the efficiency of soma/neurite separation. Relative expression levels were calculated using ΔΔCt method with Gapdh as a reference gene. See Table S7 for primer sequences.
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