Total RNA was extracted from cultured cells and insects using TRIzol Reagent (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. RT-qPCR primers were designed and tested for efficiency and specificity (Supplementary Table S2 ). First-strand cDNA was synthesized by forward primers of Tmod with total RNA as template in the reaction mixture containing M-MLV Reverse Transcriptase (Promega, USA). RT-qPCR assays were performed in Mastercycler realplex4 real-time PCR system (Eppendorf) using SYBR Green PCR Master Mix kit (Promega, USA). The succinate dehydrogenase A (SDHA) gene of N. cincticeps was used as control for each RT-qPCR assay. Quantitative analyses for relative level of gene expression were analyzed using Microsoft Excel tools.
Mastercycler realplex4 real time pcr system
Manufactured by Eppendorf
Sourced in United States, Germany
The Mastercycler realplex4 is a real-time PCR system designed for quantitative analysis of nucleic acid samples. It features four independent thermal blocks for simultaneous experiments and supports a wide range of sample volumes and plate formats. The system is capable of precise temperature control and real-time fluorescence detection, enabling accurate quantification of gene expression and other molecular targets.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
M mlv reverse transcriptase, by Promega (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix kit, by Promega (1 mentions)
Gotaq qpcr master mix kit, by Promega (1 mentions)
Sybr reagent, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Cdna synthesis kit, by Takara Bio (1 mentions)
Rnasimple total rna kit, by Tiangen Biotech (1 mentions)
Rna extraction and purification kit, by Sangon (1 mentions)
Faststart essential dna green master, by Roche (1 mentions)
5 protocols using mastercycler realplex4 real time pcr system
1
Real-Time qPCR Gene Expression Analysis
2
Quantifying RGDV Acquisition and Gene Expression in Leafhopper
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Cultured R. dorsalis cells in a monolayer with 80% confluency were inoculated with RGDV at a MOI of 1.0 for 2 h. His-Mg buffer-treated cells served as controls. At 48 hpi, the cells were collected at the same time. For viral acquisition by insects, about 500 nonviruliferous second instar nymphs of R. dorsalis were fed on RGDV-infected rice plants for 2 days, then transferred to healthy rice seedling. From 6 to 14 days padp, 30 leafhoppers were daily collected at the same time.
Total RNA was extracted from cells or insects using TRIzol Reagent (Thermo) according to the manufacturer’s instructions. For synthesizing first-strand cDNA, total RNA was primed with oligo-dT primer and reverse transcribed with M-MLV Reverse Transcriptase (Promega). The qPCR assays were performed in a Mastercycler Realplex4 real-time PCR system (Eppendorf) using GoTaq qPCR Master Mix kit (Promega) with efficient and specific primers (S1 Table ). For relative quantitation, the transcriptional level of the actin gene from the leafhopper was used as the control for each qPCR assay. Relative levels of genes were qualitatively analyzed using the 2−ΔΔCt method. For absolute quantification, the number of RGDV P8 gene copies and CASP2L and IAP gene copies were calculated as the log of the copy number/μg insect RNA based on a standard curve of the RGDV P8 gene, CASP2L gene and IAP gene, respectively.
Total RNA was extracted from cells or insects using TRIzol Reagent (Thermo) according to the manufacturer’s instructions. For synthesizing first-strand cDNA, total RNA was primed with oligo-dT primer and reverse transcribed with M-MLV Reverse Transcriptase (Promega). The qPCR assays were performed in a Mastercycler Realplex4 real-time PCR system (Eppendorf) using GoTaq qPCR Master Mix kit (Promega) with efficient and specific primers (
3
Quantitative Real-Time PCR Analysis
4
Kidney RNA Extraction and qRT-PCR
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RNA was extracted from kidneys using RNAsimple Total RNA Kit (Tiangen Biotech). Total RNA was reverse transcribed using a cDNA Synthesis Kit (Takara) to acquire cDNA. The sequences for the specific primers used are shown in Table 1 . Real-time polymerase chain reaction (PCR) was conducted using a Realplex4 Mastercycler Real Time PCR System (Eppendorf) and a SYBR® Premix Ex Taq™ Kit (TaKaRa), under universal cycling conditions (95 °C for 30 sec, 95 °C for 5 sec, and 60 °C for 30 sec, 40 cycles). The products were confirmed by appropriate size by agarose gel electrophoresis. The samples were assayed in triplicate. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control to normalize the transcript levels, and data were analyzed according to the comparative threshold cycle method.
5
Quantitative Analysis of Sesame Gene Expression
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The gene sequences for these proteins were obtained from the genome database of Yuzhi 11 (data unpublished) and quantitative real-time PCR (qRT-PCR) analysis. Total RNA was extracted from sesame leaves of all three phenotypes with an RNA extraction and purification kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The total RNA was reverse transcribed into cDNA with a Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Vilnius, Lithuania). qRT-PCR was performed with FastStart Essential DNA Green Master (Roche, Mannheim, Germany) using the Realplex 4 MasterCycler real-time PCR System (Eppendorf AG, Hamburg, Germany). qRT-PCR was conducted in accordance with the manufacturer’s instructions. The reaction conditions were as follows: 1 cycle of 50 °C for 2 min; 1 cycle of 95 °C for 10 min; 40 cycles of 95 °C for 15 s; 40 cycles of 60 °C for 20 s; and 40 cycles of 72 °C for 20 s. Three independent biological replicates per sample were performed. SiTUB was used as an internal reference gene to normalize the relative gene expression (Wei et al. 2013 (link)). The relative gene expression was calculated using the 2−∆∆Ct method (Livak and Schmittgen 2001 (link)). The primer sequences of the genes of DEPs for qRT-PCR are shown in Table 1S.
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