Validations of microarray results for chosen genes were done by quantitative real time PCR (Roche) according to manufacturer instructions. Every cDNA sample was amplified in triplicates and to check specific amplification, no-template control was also included. Further, only those samples were included in data analysis, which shows single peak in melting curve. Relative fold change in RNA expression was computed by 2−∆∆Ct method92 (link). The reference control gene used was PF3D7_1218600 (arginyl-tRNA synthetase).
Quantitative real time pcr
Manufactured by Roche
Sourced in United States
Quantitative real-time PCR is a laboratory instrument used for the amplification and detection of specific DNA or RNA sequences. It allows for the quantification of target nucleic acids by monitoring the progress of the amplification reaction in real-time. The instrument precisely measures the amount of amplified product during each cycle of the PCR process, providing accurate and reliable data for various applications, such as gene expression analysis, pathogen detection, and genetic diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Xse204 balance, by Mettler Toledo (1 mentions)
Nanophotometer n60, by Implen (1 mentions)
Rnaeasetm kit, by Beyotime (1 mentions)
Hiscript 3 all in one rt supermix perfect for qpcr kit, by Vazyme (1 mentions)
Faststart essential dna green master, by Roche (1 mentions)
32p dctp, by PerkinElmer (1 mentions)
Faststart universal sybr green master mix, by Roche (1 mentions)
4 protocols using quantitative real time pcr
1
Validating Microarray Data by qRT-PCR
2
Analytical Techniques and Instrumentation
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XSE204 balance was provided by Mettler-Toledo (Greinfesee, Switzerland). A11 basic ultra-turrax and MS3 vortex mixer were purchased from IKA (Staufen, Germany). Automatic horizontal shaker was provided by Hannuo Instruments Co., Ltd. (Shanghai, China). The precision vertical thermostatic light oscillation incubator was provided by Laibo Technology Co., Ltd. (Tianjin, China). JSM-6390 LV scanning electron microscopy was purchased from Japan Electronics Co., Ltd. (Tokyo, Japan). U-LH100-3 fluorescence microscopy was acquired from Yonke Instruments Co., Ltd. (Shanghai, China). N60 nanophotometer was provided by Implen Gmbh (Munich, Germany). Quantitative real-time PCR was purchased from Roche Diagnostics GmbH (Mannheim, Germany).
3
Parbendazole Treatment in HN6 Cells
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HN6 cells were seeded in a 6-well plate at a density of 1.5 × 105 cells per well, treated with 200 nmol/L parbendazole for 24 h and then collected. RNA was extracted using RNAeaseTM kit (Cat# R0026, Beyotime), and the reverse transcription reaction was completed using HiScript III All-in-one RT SuperMix Perfect for qPCR kit (Cat# R333, Vazyme), fluorescence quantitative analysis was performed on a Quantitative Real-time PCR (LC96, Roche) with FastStart Essential DNA Green Master (Cat# 06402712001, Roche). GAPDH and RPLPO were defined as internal reference. The primer sequence can be found in supplemental information (Table S6 ).
4
Quantitative Analysis of Serotonin mRNA
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Total RNA was isolated by using TRI reagent according to the manufacturer's instructions. About 10 mg of total RNA was sizeseparated by 1% agarose gel electrophoresis containing 0.66 M formaldehyde, transferred to nylon membranes (Pall Corporation, Port Washington, NY, USA), and hybridized with a randomly primed probe labeled with [ 32 P]dCTP (PerkinElmer). For detection of reporter mRNA, rat serotonin N-acetyltransferase (NAT)-coding region was used as probe. Radioactivity was analyzed by autoradiography.
Quantitative real-time reverse-transcription (RT) PCR Total RNA was isolated by using TRI reagent and reverse transcribed using oligo-dT and ImProm-II reverse-transcription system (Promega) according to the manufacturer's instructions. The amount of mRNA was analyzed by quantitative real-time PCR (Applied Biosystems, Foster City, CA, USA) with the FastStart Universal SYBR Green Master Mix (Roche Diagnostics). A comparative C t method was used for quantification.
Quantitative real-time reverse-transcription (RT) PCR Total RNA was isolated by using TRI reagent and reverse transcribed using oligo-dT and ImProm-II reverse-transcription system (Promega) according to the manufacturer's instructions. The amount of mRNA was analyzed by quantitative real-time PCR (Applied Biosystems, Foster City, CA, USA) with the FastStart Universal SYBR Green Master Mix (Roche Diagnostics). A comparative C t method was used for quantification.
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