The expression profile of VvSWEET genes in all the studied samples (control and infected grape berries and control and Botrytis-elicited suspension cell cultures) was analyzed by real-time qPCR performed using cDNAs obtained from RNAs extracted from each of the three composite pools of grape berry samples that constitute the three biological replicates of each condition in this study. Real-time qPCR was performed with Xpert Fast SYBR Blue (GRISP) using 1 µL of diluted cDNA (1:10) in a total of 10 µL of reaction mixture per well. For reference genes, VvACT1 (actin) and VvGAPDH (glyceraldehyde-3-phosphate dehydrogenase) were used, as they are considered extremely adequate reference genes for gene expression normalization purposes in qPCR analyses in grapevine (Reid et al., 2006 (link)). Specific primers used for each studied gene are listed in the Supplementary Table 1
Cfx manager 2
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Sourced in United States, Italy
The CFX Manager 2.1 software is a comprehensive data analysis platform developed by Bio-Rad for use with their real-time PCR detection systems. The software provides users with tools for managing experiments, analyzing data, and generating reports. Its core function is to facilitate the analysis and interpretation of real-time PCR data.
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Lab products found in correlation
Cfx96 real time system, by Bio-Rad (17 mentions)
Ssofast evagreen supermix, by Bio-Rad (11 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (11 mentions)
Iscript cdna synthesis kit, by Bio-Rad (10 mentions)
Trizol reagent, by Thermo Fisher Scientific (9 mentions)
Iq sybr green supermix, by Bio-Rad (8 mentions)
Cfx96 real time pcr system, by Bio-Rad (8 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (8 mentions)
Sybr green supermix, by Bio-Rad (8 mentions)
Rneasy mini kit, by Qiagen (7 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (7 mentions)
Rapid plant rna extraction kit, by Aidlab (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Cfx96, by Bio-Rad (5 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (5 mentions)
C1000 thermal cycler, by Bio-Rad (4 mentions)
Cfx connect real time system, by Bio-Rad (4 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Beacon designer software, by Premier Biosoft (4 mentions)
Rneasy plus mini kit, by Qiagen (4 mentions)
130 protocols using cfx manager 2
1
Grape SWEET Gene Expression Profiling
2
Quantitative RT-PCR for Gene Expression Analysis
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qRT-PCR was performed with a KAPA SYBR FAST qPCR kit (Kapa Biosystems) in the CFX96 Real-Time system (Bio-Rad Laboratories). Thermal cycling conditions were as follows: denaturation (95°C for 3 min), followed by 35 cycles of denaturation (95°C for 5 min), annealing, and extension (60°C for 30 min). Fluorescence detection and data analysis were performed with CFX Manager 2.0 (Bio-Rad Laboratories). Experiments were performed in triplicate, with RNF111 (Entrez Gene ID 54778 [OMIM 605840]) as the reference gene for the normalization of expression data (Bustin et al., 2009 (link)) and COPS5 (Entrez Gene ID 10987 [OMIM 604850]) as a secondary housekeeping gene. The primers used are also listed in Table S4. For qRT-PCR after transduction, see Stimulation and qRT-PCR.
3
Quantifying Cotton Fiber Synthase Genes
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Quantitative RT-PCR (qRT-PCR) was employed to detect the expression levels of predominant PA synthase genes in each 10 brown and white fiber RILs. The investigated genes and corresponding primers were listed in table 1 . The histone 3 and elongation initiation factor 5 genes from cotton were amplified as RNA standard [23] (link), [24] (link). PCRs were performed on a CFX96™ real-time PCR detection system with SYB Green supermix (Bio-Rad, CA, USA). The thermocycling parameters were as follows: 95°C, 2 min, 40 cycles of 95°C, 10 s and 57°C, 20 s. A standard melting curve was added to monitor the specificity of PCR products. The reactions were duplicated for 3 times and data were analyzed using the software Bio-Rad CFX Manager 2.0 provided by manufacturer.
4
Protocol for RNA Extraction and qRT-PCR
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Total RNA from LV tissue was extracted using TRI Reagent® (Sigma-Aldrich), as described.27 (link),30 (link) From total RNA, 0.5 μg was reverse transcribed to produce cDNA using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA). cDNA was subjected to quantitative real-time polymerase chain reaction (PCR) using the C1000 Thermal Cycler CFX384 Real-Time PCR Detection System (Bio-Rad) using iQ SYBR green super mix (Bio-Rad). Quantification of gene expression levels was performed with Bio-Rad CFX Manager 2.0. All gene expression levels were normalised to the reference gene 36B4 and were presented as fold change to the control group. Primer sequences that were used for quantitative PCR analyses are displayed in Supplementary material online , Table S3 .
5
Quantitative Gene Expression Analysis
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After RNA-seq, qRT-PCR was performed by SYBR Premix Ex Taq II (Tli RNaseH Plus) in a volume of 10 μL, which contained 5 μL of SYBR Green Master Mix, 200 ng cDNA template, and 0.5 μM of each of the forward and reverse primers. The qRT-PCR amplification conditions were as follows: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s in Bio-Rad CFX manager 2.0. The relative expression levels were estimated from the threshold of PCR cycle with 2–ΔΔCt method (Livak and Schmittgen, 2001 (link)). The values from three independent biological replicates and three technical replicates were averaged.
6
Precision Assessment of Quantitative PCR
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For the assessment of precision of our method and comparison with the classical calibration curve method, we produced 16 replicas of a 6-step dilution series. We provide the detailed pipetting layout in Additional file 1 : Figure S2. Two datasets were generated from this experiment and processed using Bio-Rad CFX Manager 2.0 (2.0.885.0923). Additional file 2 : Dataset 1 consists of relative fluorescence data obtained from the aforementioned experiment: 6 serial dilution wells × 16 replicas = 96 wells. Fluorescence data in Additional file 2 : Dataset 1 are expressed as RFU (Relative Fluorescence Units) which is a term specific to Bio-Rad software. It is important to note that, since our goal was to improve the accuracy of the classical calibration curve, all RFU values were taken as already processed by Bio-Rad software with the same settings that were applied to the generation of Cq values, as follows: Baseline Setting set to Baseline Subtracted Curve Fit, Cq Determination Mode set to Single Threshold. Additional file 3 : Dataset 2 contains automatically generated Cq values corresponding to Additional file 2 : Dataset 1. The threshold was automatically set at 31.07 by the Bio-Rad software.
7
Analyzing Liver Transcript Levels using Real-Time qPCR
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Samples of liver tissue (100 mg) frozen in liquid nitrogen were ground and subsequently used for extraction of total RNA with Trizol according to instructions of the vendor (Invitrogen; San Diego, CA, USA). The cDNA of patient liver samples was prepared and real-time qPCR was performed as described previously [6 (link)]. The primers used in this study are given in S1 Table . OSTα, OSTβ, GAPDH and β-actin primers were used as described [6 (link), 23 (link)]. GAPDH and β-actin were used as references for normalizing data, and real-time PCR amplification efficiency of target genes was considered, when using CFX manager 2.0 for data analysis (Bio-Rad).
8
Quantifying Oxidative Stress and Notch Signaling
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Oxidative stress arrays were purchased from Qiagen. RNA was reverse transcribed to cDNA with the RT2 First Strand Synthesis kit (Qiagen). The cDNA was combined with SYBR Safe Mastermix (Qiagen) and aliquoted across the array plate. All array plate qRT‐PCR was performed using the C1000 Thermal Cycler and CFX96 Real‐Time System (BioRad, Hercules, CA. USA) under the RT2 Array qRT‐PCR protocol; –10 min, 39 cycles of 95 °C for 10 s, 60 °C for 1 min. Data was assimilated using the CFX Manager 2.0 (BioRad) and analysed using the Qiagen online Data Analysis Center (http://www.qiagen.com/gb/shop/genes-and-pathways/data-analysis-center-overview-page/ ). Gene expression scatterplots generated in the software were of the Log102 - Δ Δ C t
9
Quantitative Real-Time PCR Protocol
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All RNA extractions (both for individual qRT‐PCRs and for the RT2 Profiler qRT‐PCR Arrays) were performed on cell pellets previously stored at −80 °C or by direct in‐plate lysis (addition of complete RLT buffer followed by storage at −80 °C overnight) using the RNeasy Micro Kit (Qiagen) following the manufacturer's instructions for the animal cell protocol. Genomic DNA contamination was removed by on‐column application of the RNase‐free DNase Set (Qiagen). cDNA was synthesised using Superscript III cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA), with reactions placed in GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA). Taqman qRT‐PCR was then performed in FrameStar 96 qRT‐PCR plates (4titude, Dorking, UK). Input cDNA was standardised at 30 ng per well. The mastermix used was TaqMan Fast Universal Master Mix (Applied Biosystems). Probes used in experiments are provided in Table S4 . qRT‐PCR was performed using the C1000 Thermal Cycler and CFX96 Real‐Time System (BioRad) under the following protocol; 95 °C – 10 min, 39 cycles; 95 °C for 10 s, 60 °C for 1 min, 4 °C Hold. Data was assimilated using the CFX Manager 2.0 (BioRad) and analysed using the 2 - Δ Δ C t prism 7 (GraphPad).
10
Characterization of JAZF1 Expression
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Total liver, muscle, and adipose tissue proteins were extracted and used for SDS-PAGE at 80 V for 30 min and 120 V for 90 min. After transfer to a membrane (wet), a primary antibody (rabbit anti-mouse JAZF1 polyclonal antibody; Abcam, Cambridge, UK) was added and incubated at 4°C in a refrigerator overnight, which was followed by washing. The secondary antibody (HRP-sheep-rabbit-IgG; Abcam) was diluted (1: 1000) and incubated at room temperature for 70–80 min. After washing, Quantity One software (BIO-RAD, Hercules, CA, USA) was used for imaging and IOD values were calculated. By calculating the ratio of each value to the IOD value of internal controls, the relative expression of the target protein was determined.
For RT-PCR, mRNA extraction and reverse transcription were performed using the TAKARA mRNA Extraction and Reverse Transcription Kit. The reaction conditions were as follows: 37°C for 15 min, 85°C for 5 s, and 4°C for 20 min. PCR was performed immediately using a PCR instrument (Hangzhou Anjes) with the following primers for JAZF1: 5′-CACGCCGAGAACAGGAAT-3′ and 5′-GTGCTGCTGCGGAATGAA-3′. β-actin was used as the internal reference. The specificity and relative amplification efficiency were evaluated using Bio-Rad CFX Manager 2.0.
For RT-PCR, mRNA extraction and reverse transcription were performed using the TAKARA mRNA Extraction and Reverse Transcription Kit. The reaction conditions were as follows: 37°C for 15 min, 85°C for 5 s, and 4°C for 20 min. PCR was performed immediately using a PCR instrument (Hangzhou Anjes) with the following primers for JAZF1: 5′-CACGCCGAGAACAGGAAT-3′ and 5′-GTGCTGCTGCGGAATGAA-3′. β-actin was used as the internal reference. The specificity and relative amplification efficiency were evaluated using Bio-Rad CFX Manager 2.0.
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