Primers for avp were designed and optimised by Primerdesign Ltd. The primer sequences were: avp forward: 5′-CTGCCTGCTACATCCAGAACT-3′, avp reverse: 5′-CACACGACATACACTGTCTGATG-3′. The sequences of the primers for oxt, th, th2, tph1a, tph1b, tph2, avpr1aa, avpr1ab were taken from33 (link) and purchased from Sigma. RNA was extracted from the whole brain using the GeneEluteTM Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) followed by a DNase treatment with Turbo DNase (Ambion). The quality and quantity of RNA was assessed using a Nanodrop 2000 (Thermo Scientific). cDNA was synthesised from 0.5 µg of RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). Real-time PCR was performed on 8 whole brains per genotype with three replicates for each brain using a CFX ConnectTM Real-Time System machine (BIORAD) and the SensiFASTTM SYBR No-ROX Mix (Bioline). The PCR conditions were 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s and 72 °C for 30 s. Results were normalised to the expression level of the housekeeping gene rpl13. The relative expression of the genes was calculated using the comparative 2−ΔΔCt method as described in97 (link).
Cfx connect real time system machine
Manufactured by Bio-Rad
Sourced in United States
The CFX Connect Real-Time System is a laboratory instrument designed for real-time PCR (polymerase chain reaction) analysis. It provides a platform for performing quantitative gene expression analysis, genotyping, and other real-time PCR applications. The system includes a thermal cycler, optical detection system, and associated software for data acquisition and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Qx200 droplet digital pcr ddpcr system, by Bio-Rad (2 mentions)
Gene elute mammalian total rna miniprep kit, by Merck Group (1 mentions)
Sensifast sybr no rox mix, by Meridian Bioscience (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
Ez 10 spin column plant rna mini preps kit, by Bio Basic (1 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
Itaq universal sybr green 1 step reaction mix, by Bio-Rad (1 mentions)
Evagreen dye, by Biotium (1 mentions)
Phusion hot start flex, by New England Biolabs (1 mentions)
Omniscript, by Qiagen (1 mentions)
Rneasy extraction kit, by Qiagen (1 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Omniscript reverse transcription kit, by Qiagen (1 mentions)
M mlv rt, by Promega (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
13 protocols using cfx connect real time system machine
1
Optimized Real-time PCR for Brain Gene Expression
2
Plant RNA Extraction and qRT-PCR
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Total RNA was extracted from 50 mg plant tissue, either 12-day-old 1/2 MS medium-grown seedlings or 4-week-old soil-grown plants using the EZ-10 Spin Column Plant RNA Mini-Preps Kit (Bio Basic, Canada). 2 µg RNA was reversely transcribed to cDNA using Easy ScriptTM reverse transcriptase (ABM, Canada). 50 ng cDNA was added as a template in a 10 µl reaction on a Bio-Rad CFX ConnectTM Real-Time system machine. Real-time PCR was conducted to quantify the relative expression level of the target genes. ACTIN1/7 was used to normalize the expression value.
3
qRT-PCR expression analysis protocol
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Total RNA was extracted using the Qiagen RNAeasy kit with on-column DNase digestion according to the manufacturer’s instructions. qRT-PCR was performed on a Bio-Rad CFX Connect Real-Time System machine using iTaq Universal SYBR Green 1 Step Reaction Mix (Bio-Rad). Expression was normalized against the RPL19 house keeper gene. PCR conditions and primer sequences were as described previously [3 (link)].
4
Quantitative real-time PCR for sEVs
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After antibody tagging and capturing of the sEVs on beads (see section 2.5), approximately one million beads (about 1/10 carrying sEVs) were used for quantitative real‐time PCR in bulk. In each experiment, a negative control, in which sEVs were excluded during incubation with antibodies, was used to investigate background levels from barcoded antibodies. A second negative control containing only water was also used. The PCR mixture (50 μl) contained 1× Phusion Hot Start Flex (New England BioLabs; Cat. No. M0536L), 0.2 μM of H3 and H4’ primers (Table S2 ) and EvaGreen Dye (Biotium; Cat. No. 31000). Amplification was done using the following protocol: 5 min at 95°C; 40 cycles of 30 s at 94°C (ramp speed 40%), 30 s at 60°C (ramp speed 60%), and 65 s at 72°C (ramp speed 30%). The real‐time amplification reactions were performed on a CFX Connect Real‐Time System machine (Bio‐Rad).
5
Quantitative Detection of Histidine Decarboxylase Gene
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The isolates were tested for the presence of the histidine decarboxylase (hdcA) gene through qPCR performed using a CFX Connect Real-Time System machine (BioRad, Hercules, CA, USA), following the cycling conditions and primers previously described by Belleggia et al. [29 (link)] for the amplification of a 174 bp fragment of the hdcA gene [30 (link)]. The positive strain Lactobacillus parabuchneri DSM 5987 was used to create the standard curve. The analysis was performed in triplicate for each isolate, together with a blank. The results were expressed as the presence (+) or absence (−) of the target gene in the bacterial DNA samples.
6
Medicago Seedling Root RNA Isolation and Analysis
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RNA was isolated from 7-day-old Medicago seedling roots with an RNeasy Extraction kit (Qiagen). Then, cDNA was generated from total RNA (500 ng) by reverse transcription using Omniscript (Qiagen), according to the provided protocol. For material treated with phenolic compounds, droplet digital PCR was performed with the QX200 Droplet Digital PCR (ddPCR™) System (Bio-Rad) using EvaGreen. For material treated with the fungal elicitor, real-time analyses were performed in a CFX Connect Real-Time System machine (Bio-Rad) using SYBR Green. Actin was used as a reference gene for normalization. For the real-time PCR assays, the gene expression levels were determined by the ΔΔCt method. For primer sequences, see Supplementary Table S1 , available at JXB online.
7
Antifungal Responses of A. fumigatus to 5,8-diHODE
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A. fumigatus Af293 WT spores were inoculated at 106 spores/mL, grown overnight in liquid GMM at 37 °C and 250 rpm, and treated with either 5,8-diHODE (5 μg/mL) or EtOH (0.005%) for 30 min and 120 min. Four biological replicates were included for each condition. At 30 min and 120 min post treatment, total fungal biomass was harvested, flash-frozen in liquid nitrogen, and lyophilized. The total RNA was extracted using QIAzol Lysis Reagent (Qiagen) according to the manufacturer’s instructions with additional phenol:chloroform:isoamylalcohol (24:1:1) extraction step before RNA precipitation. For quantitative RT-PCR, RNA was digested with DNase I (New England Biolabs) and reverse-transcribed using iScript cDNA synthesis kit (Biorad). Quantitative PCR was performed using iQ SYBR Green Supermix (Bio-Rad) following the manufacture’s cycle conditions in the CFX Connect Real-Time System machine (Bio-Rad), with 12.5 ng input cDNA and primers listed in Supplementary Table 4 . The expression level of each gene was normalized to act1 expression level of the respective sample using the 2−∆∆CT (Livak) method54 (link).
8
Molecular Characterization of Mycorrhizal Symbiosis
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Total RNA of the collected samples were extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The cDNA was then synthesized with an Omniscript Reverse Transcription (RT) Kit (Qiagen) or Moloney Murine Leukemia Virus Reverse Transcriptase Reverse Transcriptase (M-MLV RT) (Promega, Madison, Wisconsin, USA). Quantitative PCR analyses for MtABCG43, MtABCG44, MtABCG59, MtCCD7, and MtCCD8 were performed in a CFX Connect Real-Time System machine (BioRad, Hercules, CA, USA) using iTaq Universal SYBR Green supermix (BioRad) with at least three biological replicates each with three technical repeats. Quantitative PCR analyses for the AM-marker genes (MtBCP1 and MtPT4) were performed in a 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, Massachusetts, USA) using SYBR Green PCR Master Mix (Applied Biosystems) with four biological replicates each with two technical repeats. Expression levels were normalized to Mtactin and calculated with the ΔΔCt method. For primer sequences, see Supplementary Table 1
9
RNA Extraction and Gene Expression Analysis
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RNA was isolated from plant material with an RNeasy Extraction kit. Genomic DNA was removed by on‐column DNase treatment. Total RNA (500 ng) was converted to cDNA with Omniscript reverse transcriptase (Qiagen, Hilden, Germany) following the manufacturer's protocol. Droplet digital PCR was performed with the QX200 Droplet Digital PCR (ddPCR™) System (Bio‐Rad, Hercules, CA, USA) using EvaGreen. RT‐PCR was performed in a CFX Connect Real‐Time System machine (Bio‐Rad) using SYBR Green. Actin was used as a reference gene for normalization, and the gene expression levels were determined by the ΔΔCt method. For primer sequences, see Table S2 .
10
Quantitative RT-qPCR for Viral Gene Expression
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Total cellular RNA was isolated using the RNeasy mini kit followed by mRNA isolation using the Oligotex mRNA Mini Kit (both from Qiagen). qPCR assay was performed with Blaze Taq One-step SYBR Green RT-qPCR kit (Genecopoeia) using 5 ng total mRNA for all the samples with the following direct and reverse primers, respectively: NP (5′-CACCAGAAGACATCACCGATAC, 5′-CGACTCATCTGCAGTCTCATAC), HA (5′-TTTCTCGACGTGTGGACTTAC, 5′-CTGGAGCCGGACTTTATCATAG), actin (5′-ACAGAGCCTCGCCTTTG, 5′-CCTTGCACATGCCGGAG). The amplification was performed on a CFX Connect Real-Time System machine and the data were collected with CFX Maestro software (Bio-Rad). The level of HA mRNA relative to that of NP normalized to the control sample was calculated using the ΔΔCt method.
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