Total RNA was extracted using a QIAamp Viral RNA Mini Kit (QIAGEN, Venlo, The Netherlands) according to the manufacturer’s instructions as described previously [36 (link)]. Subsequently, cDNA synthesis was performed using total RNA and a SensiFAST™ SYBR Lo-ROX One Step Kit (Meridian Bioscience, OH, USA). RT-qPCR was carried out as described previously [37 (link)]. The master mix, comprising 10 µl of 2× SensiFAST™ SYBR Lo-ROX One Step Mix, 0.8 µL of 10 µM forward and reverse primers, 0.2 µL of reverse transcriptase, 0.4 µL of Ribosafe RNase inhibitor, 5.8 of µL water, and 2 µL of RNA template, had a total volume of 20 µL. The primers IAV PB1-F (F:5'-GGCCCTTCAGTTGTTCATC-3') and IAV PB1-R (3'-GTGTAAGGACTTCAGACG-5') were used in the reaction. The reaction was performed using a LineGene 9600 Plus Real-time PCR detection system (Bioer technology, Hangzhou, CN).
Sensifast sybr lo rox one step kit
Manufactured by Meridian Bioscience
Sourced in United States
The SensiFAST SYBR Lo-ROX One-Step Kit is a reagent kit designed for reverse transcription and real-time PCR amplification of RNA targets. The kit provides a one-step, sensitive, and reproducible solution for gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
7500 fast real time pcr system, by Meridian Bioscience (3 mentions)
Quantitect primer assay, by Qiagen (3 mentions)
Qiaamp viral rna mini kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Linegene 9600 plus real time pcr detection system, by Bioer (1 mentions)
Total rna mini kit, by Geneaid (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Ddit3, by Qiagen (1 mentions)
Folin ciocalteau reagent, by Merck Group (1 mentions)
Butylated hydroxytoluene, by Merck Group (1 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (1 mentions)
Mytaq red mix kit, by Meridian Bioscience (1 mentions)
Easy spin dna free total rna extraction kit, by iNtRON Biotechnology (1 mentions)
Rat il 6 elisa kit, by RayBiotech (1 mentions)
9 protocols using sensifast sybr lo rox one step kit
1
RNA Extraction and RT-qPCR for Influenza Virus
2
Quantifying Influenza Genome Copy Numbers
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The reduction in genome copy numbers was confirmed by RT-qPCR quantification of influenza treated with extracts as previously described [38 (link),39 (link)]. RNA was extracted from the supernatant of each group of antiviral tests using the QIAamp Viral RNA Mini kit following the handbook’s instructions (QIAGEN, Hilden, Germany). RT-qPCR was performed through the SensiFAST SYBR Lo-ROX One-Step kit (Meridian Bioscience, Cincinnati, OH, USA) using the following primer pairs: IAV PB1 (Forward, 5′-GGC CCT TCA GTT CAT C-3′, Reverse 5′-GCA GAC TTC AGG AAT GTG-3′). The reaction mixture used consists of 2 µL of RNA template, 10 µL of 2× One-step mix, 1.6 µL of primer (forward + reverse), 0.2 µL of reverse transcriptase, and 0.4 µL of RNase inhibitor. Water was filled up to the final total reaction volume for each sample of 20 µL. The cycling condition was performed as follows: 45 °C for 30 m and 95 °C for 10 m, and then the three-step reaction of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 15 s was repeated 40 times.
3
Quantitative Gene Expression Analysis in Drosophila
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This assay was performed as previously described [82 (link)]. Total RNA was extracted from 30 fly heads per sample using TRIzol (Ambion), and quantified by spectrophotometric analysis (Nanodrop, Thermo Scientific). Quantitative real-time PCR with reverse transcription (RT–qPCR) was performed with a real-time cycler (Applied Biosystems 7500, Fast Real-Time PCR Systems) using the SensiFAST SYBR Lo-ROX One-Step Kit (Bioline). Fold change was calculated using the comparative Ct method. For RT–qPCR we measured the coefficient of variation (CV) of the technical replicates and excluded from statistical analysis any samples with CV > 3%. Gene-specific primers were designed with FlyPrimerBank (DRSC FlyPrimerBank (flyrnai.org)), and subsequently obtained from Sigma:
Get4: forward, 5′-TACGGCGCAGAAACGCTATC-3′,
reverse 5′-GCTTTCCTGTTCCTTGGCAATAA-3′.
rp49 was used as the housekeeping gene:
forward, 5′-TGTCCTTCCAGCTTCAAGATGACCATC-3′,
reverse 5′-CTTGGGCTTGCGCCATTTGTG-3′.
Get4: forward, 5′-TACGGCGCAGAAACGCTATC-3′,
reverse 5′-GCTTTCCTGTTCCTTGGCAATAA-3′.
rp49 was used as the housekeeping gene:
forward, 5′-TGTCCTTCCAGCTTCAAGATGACCATC-3′,
reverse 5′-CTTGGGCTTGCGCCATTTGTG-3′.
4
H9N2 Virus Binding Inhibition Assay
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The H9N2 virus binding inhibition test on red blood cells was carried out in six repetitions with graded sialidase concentrations (0 U/mL, 0.032 U/mL, 0.064 U/mL, 0.129 U/mL and 0.258 U/mL). A total of 500 μl of graded sialidase concentration was added to 500 μl of 1% concentration of chicken and rabbit red blood cells and then incubated at 37 °C for 2 h. After incubation, 500 μl 9 log 2 HAU of avian influenza H9N2 virus was poured into a mixture of sialidase and red blood cells and then incubated for 30 min at room temperature. Then centrifuged at 5000 rpm for 5 min. The supernatant was discarded and the pellet in the form of red blood cells was extracted by RNA using the Total RNA Mini Kit (Geneaid) and then followed by qRT-PCR to count the number of RNA copies of the virus that were still bound to the red blood cells. The qRTPCR process was carried out using the Sensifast SYBR Lo-ROX One-Step Kit (Bioline) kit according to the protocol for use.
5
RNA Extraction and qRT-PCR Analysis of Drosophila S2 Cells
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Total RNA was isolated from S2 cells (approximately 0.5 × 105) with Tri reagent (Sigma-Aldrich). 100ng RNA was analyzed by using SensiFAST SYBR Lo-ROX One-Step Kit (Bioline) in StepOnePlus System (Life Technologies). The primers used for detection were: Ser: CATAACAACCTGTAGCGCGC, TCGCCGAATCCTTGTCGAAA; Shi: ATTCGCAAGGGTCACATGGT, ACCATCCAACGGCAGCATAA; Rab5: CATCGAACTCTACGACGCGA, CCTGGGTCAAGGAACTGCAT; Rab7: AGGGCATCAACGTGGAGATG, CGATTGTTTTGCGAGCCCAA; Mam: TATCAGCACAGCTTCTGGGAC, ACGCGGAGAGGTGTTAGGA; Dor: ACATGAAGCTGGCCAAGGAA, TGCGTAAGAGATCGCACTCC; Kuz: GAAGGCATTGCTGACCACGA, CGTCGAACTTTGTGTTGCGG; Notch: GCAAGTGCCCCAAAGGTTTC, CAGGTGTAGTCCGAGATGCC; CHC: TGACATGAACGATGCCACCA, TGCAACGTCTTTTGCGCTTT; Synj: AACTAATGGATGGTGCGCGA, GGACATCAATGGCTTCCTGC; GlcT1: CTTCGCGGCATTCTTCATGG, GCTTGCAGGACTTCTTGTGC; Egh: CGTTTTGGCATGGAAAACATGAA, GTGAACTTGGACGTGGTTGC; β4galNACTB: AACAGGGCGATGCTCTTCAA, CGAATTCAGTGGCAGGAGGT; RP49RT: AGTATCTGATGCCCAACATCG, TTCCGACCAGGTTACAAGAAC.
6
Quantitative Real-Time PCR for Gene Expression
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Total RNA was extracted using the RNeasy Mini Kit (QIAGEN; for mammalian cells) or TRIzol (Ambion, Waltham, MA, USA; for Drosophila tissues) and quantified by spectrophotometric analysis. Quantitative real-time PCR with reverse transcription (qRT–PCR) was performed on a real-time cycler (Applied Biosystems, Foster City, CA, USA, 7500 Fast Real-Time PCR Systems) using the SensiFAST SYBR Lo-ROX one-Step Kit (Bioline, London, UK). Gene-specific primers were obtained from QIAGEN (QuantiTect Primer Assays) for the following genes: DDIT3 (QT00082278), dATF4 (QT00503069), CG3999 (QT00972349), CG6415 (QT00934318), Nmdmc (dm: QT00503153, hs: QT00081592), Parkin (QT00023401), Pink1 (QT01670459) and Shmt2 (dm: QT00498904, hs: QT00012754). Gene-specific primers obtained from Sigma were: hsATF4 (forward, 5′-CCCTTCACCTTCTTACAACCTC-3′ reverse, 5′-GTCTGGCTTCCTATCTCCTTCA-3′), rp49 (forward, 5′-TGTCCTTCCAGCTTCAAGATGACCATC-3′ reverse, 5′-CTTGGGCTTGCGCCATTTGTG-3′), TBP (forward, 5′-TCAAACCCAGAATTGTTCTCCTTAT-3′ reverse, 5′-CCTGAATCCCTTTAGAATAGGGTAGA-3′). rp49 and TBP were used as housekeeping genes when analysing fly and cells samples, respectively.
7
Antioxidant and Hepatoprotective Evaluation
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Folin–ciocalteau reagent, 4-hydroxycinnamic acid (4-HCA), rutin (RU), catechin, butylated hydroxytoluene (BHT), 2,2- azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS),monoisoamylDMSA, thiobarbituric acid (TBA), reduced glutathione (GSH), and 5,5`-{dithiobis-2-nitrobenzoic acid} (DTNB) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Lead acetate was purchased from ISO-CHEM, France. Alkaline phosphatase (ALP), Albumin, ALT and AST kits were obtained from Giesse Diagnostics, Italy. Urea and creatinine kits were supplied by Diamond Diagnostics, Egypt. Easy-spin™ [DNA free] total RNA extraction kit was obtained from iNtRON Biotechnology, South Korea. SensifastsyBr lo-Rox one-step kit and MyTaq Red Mix kit were purchased from Bioline (USA). Primers and 50 bP DNA ladder kit were obtained from Vivantis (USA) and Genedirx (USA), respectively. Rat IL-6 ELISA kit was purchased from RayBiotech (Norcross, USA). Other chemicals were obtained with a high grade.
8
Measuring Gene Expression via RT-qPCR
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Total RNA was extracted from 25 heads or 15–20 freshly dissected guts using TRIzol (Ambion) and quantified by spectrophotometric analysis (Nanodrop, Thermo Scientific). RT‒qPCR was performed with a real-time cycler (Applied Biosystems 75000, Fast Real-Time PCR Systems) using the SensiFAST SYBR Lo-ROX One-Step Kit (Bioline). The fold change in expression was calculated using the comparative Ct method [35 (link)]. For RT‒qPCR, we measured the coefficient of variance (CV) of the technical replicates, and any samples with a CV > 3% was excluded from statistical analysis. The gene-specific primers were Dm_PEK_2_SG QuantiTect Primer Assay (Qiagen, QT00968618), and Relish (forward, 5′-CATCAGGAGACAGAGCGTGA-3′; reverse, 5′-CCGACTTGCGGTTATTGATT-3′) and rp49 (forward, 5′-TGTCCTTCCAGCTTCAAGATGACCATC-3′; reverse, 5′-CTTGGGCTTGCGCCATTTGTG-3′) (Sigma‒Aldrich).
9
Quantitative RT-PCR for Drosophila Genes
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Total RNA was extracted using TRIzol (Ambion) and quantified by spectrophotometric analysis. Quantitative real-time PCR with reverse transcription (qRT-PCR) was performed on a real-time cycler (Applied Biosystems 7500 Fast Real-Time PCR Systems) using the SensiFAST SYBR Lo-ROX one-Step Kit (Bioline). Gene-specific primers were obtained from QIAGEN (QuantiTect Primer Assays) for the following genes: dMfn (QT00499205), Nmdmc (dm: QT00503153), Shmt2 (dm: QT00498904). Gene-specific primer rp49 (forward, TGTCCTTCCAGCTTCAAGATGACCATC; reverse, CTTGGGCTTGCGCCATTTGTG) was obtained from Sigma and used as a housekeeping gene.
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