First-strand cDNA synthesis was conducted using 5 µg of total RNA from the anthers of SW-S and SW-F at the tetrad stage with a PrimeScript RT reagent kit (Takara, Dalian, China) according to the manufacturer’s instructions. The SmActin gene (DQ243702) was employed as an internal control for qRT-PCR (Yang et al., 2010 (link)). The primers for the selected genes and SmActin gene were designed using Beacon Designer 7, and are listed in Table S1 . qRT-PCR assays were performed with SYBR Green Dye (Takara, Dalian, China) using a Bio-Rad CFX96 real-time PCR platform (Bio-Rad, Hercules, CA, USA) according to the following program: 95 °C for 5 min, followed by 40 cycles at 95 °C for 5 s and 55−63 °C (annealing temperature varied according to primers) for 30 s. All assays for a particular gene were performed three times synchronously under identical conditions, and RNA transcript fold changes were calculated using the 2−ΔΔCt method (Livak & Schmittgen, 2001 (link)).
Cfx96 real time pcr platform
Manufactured by Bio-Rad
Sourced in United States, China
The CFX96 real-time PCR platform is a thermal cycler used for quantitative real-time polymerase chain reaction (qPCR) experiments. It can detect and measure DNA or RNA sequences in real-time during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green dye, by Takara Bio (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
Primescript rt reagent kit perfect real time, by Takara Bio (2 mentions)
Chamq sybr qpcr master mix, by Vazyme (2 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr green pcr master mix kit, by Takara Bio (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Mircute mirna qpcr detection kit, by Tiangen Biotech (1 mentions)
Hiscript 2 q select rt supermix for qpcr, by Vazyme (1 mentions)
Mircute plus mirna fist strand cdna kit, by Tiangen Biotech (1 mentions)
Sybr green 2, by Takara Bio (1 mentions)
First strand cdna synthesis kit, by Takara Bio (1 mentions)
Sybr green supermix reagent, by Bio-Rad (1 mentions)
Dnase, by Thermo Fisher Scientific (1 mentions)
M mlv transcriptase, by Promega (1 mentions)
Klustercaller software, by LGC (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
22 protocols using cfx96 real time pcr platform
1
Real-time qRT-PCR for Gene Expression
2
Validating RNA-Seq Analysis via qRT-PCR
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The reliability of the RNA-Seq analysis was confirmed using qRT-PCR analysis of 10 genes believed to affect the meat quality traits and muscle carcass traits of CH and DG chicken breeds. Primers were designed by Primer-BLAST on the NCBI website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) accessed on 1 March 2022 based on the Gallus_gallus-5.0 (NCBI) and synthesized by the Kunming synthesis department of TSINGKE biological technology Co., Ltd. (Kunming, China). Table S2 , for specific primer information. Seven samples were taken from each breed to collect tissue, the same chicken that was used for transcriptome analyses was used, and each sample was repeated three times. The total RNA of the breast muscle of chickens was then isolated and extracted using a kit produced by Bao Biological Engineering (Dalian) Co., Ltd., Dalian, China, according to the manufacturer’s instructions. cDNA was synthesized using the Prime Script RT reagent kit with gDNA Eraser (Takara, Dalian, China). qRT-PCR was performed using the Bio-Rad CFX96 real-time PCR platform (Bio-Rad Laboratories. lnc, California, CA, USA) with the SYBR® Green PCR Master Mix Kit (Takara, Dalian, China). β-actin was employed as a reference gene for the normalization of gene expression levels. The fold change values were calculated using the 2-ΔΔCt method [39 (link)].
3
Validating RNA-seq Data with qRT-PCR
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Twenty-five primer pairs (Additional file 4 : Table S3) were designed to generate amplicons to validate the RNA-seq data. Aliquots of the total RNA extracted for sequencing were used for quantitative real-time PCR experiments in accordance with manufacturer’s instructions (Qiagen, Shanghai, China). Real-time assays were performed with SYBR Green Dye (Takara, Dalian, China) using a Bio-Rad CFX96 real-time PCR platform (Bio-Rad Laboratories, Hercules, CA, USA). All assays for a particular gene were performed three times synchronously under identical conditions, and RNA transcript fold changes were calculated through the 2-ΔΔCtmethod [42 (link)] with the wheat housekeeping genes Ubiq (DQ086482) and Actin (AB181911) as internal controls [13 (link),43 (link)].
4
Parvovirus B19 Detection in Plasma
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Individual plasma samples were tested by pools of 48 individuals. The samples in the B19V positive pools and the pooled plasma were tested separately with 960 µL plasma. A virus DNA/RNA kit (Beijing Wantai Biological Pharmacy Enterprise Co., Ltd, Beijing, China) was used for nucleic acid extraction according to the manufacturer’s instructions. The DNA extracts were stored at −80 °C prior to PCR analysis. B19V DNA samples were screened using human parvovirus B19V DNA diagnostic kits (PCR-fluorescence probing) (Beijing Wantai Biological Pharmacy Enterprise Co., Ltd, Beijing, China). This kit can detect all three B19V genotypes with a sensitivity of 20 IU per mL. The Q-PCR assays were performed on a Bio-Rad CFX96 real-time PCR platform (Bio-Rad Laboratories, Hercules, CA, USA).
5
Gene Expression Analysis by Real-Time qRT-PCR
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After RNA extraction, cDNA synthesis was performed using the SuperscriptIII First-strand synthesis system (Invitrogen, Shanghai, China) as the manufacturer’s instructions. Real-time PCR was performed using SYBR Premix Ex Taq (Takara, Dalian, China) with a Bio-Rad CFX96 real-time PCR platform (Bio-Rad, Hercules, CA, United States). GAPDH transcripts were used as the internal control (Løvdal and Lillo, 2009 (link)). The qRT-PCR primers used in this study were listed in Supplementary Table 1 . The efficiency of primers was calculated before carrying out the qRT-PCR reaction, all 10 gene primers of good quality with a PCR efficiency (in r-squared value) > 0.9 (Supplementary Table 1 ). The qRT-PCR was performed with three technical replicates.
6
Quantitative Real-Time RT-PCR Analysis
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For quantitative real-time RT-PCR, the reference genes 18S (GenBank No. AY049040) and actin (GenBank No. AB181911) (Yamada et al., 2009; Wang et al., 2010) were used (Table 1); the PrimeScript Perfect Real-Time RT Reagent Kit (TaKaRa, China) was used for the reactions. The primers were designed using the Primer Express 3.0 software for the HGF2, HDB2, and HCG4 genes (Table 1). PCR was conducted in triplicate and normalized to 18S and actin. Real-time expression assays were performed with SYBR Green dye (TaKaRa) using the BIO-RAD CFX-96 real-time PCR platform (Bio-Rad). Fold-changes of RNA transcripts were calculated via the 2 -ΔΔCt method (Livak and Schmittgen, 2001) .
7
Quantitative Analysis of miRNAs and Target Genes
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Total RNA was extracted from previously frozen plant tissues using RNAsimple Total RNA kit. For each examined miRNA, 1 μg of total RNA was used as the template for reverse transcription using the miRcute Plus miRNA Fist-Strand cDNA kit (TIANGEN, Beijing, China); the U6 gene was used as the internal control. Reverse transcription was performed using the following conditions: 42 °C for 60 min, 95 °C for 3 min, hold at 4 °C. For target genes, 1 μg of total RNA was used for synthesis with HiScript II Q Select RT SuperMix for qPCR (Vazyme, Nanjing, China) and an oligo (dT) 23 primer, and the gene UBQ was used as an internal control. Reverse transcription was performed using the following conditions: 50 °C for 15 min, 85 °C for 5 s, hold at 4 °C. QPCR was performed on the Bio-Rad CFX96 Real-time PCR platform. The MiRcute miRNA qPCR Detection kit (TIANGEN, Beijing, China) and ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China), respectively, were used for the RT-qPCR assays according to the manufacturer’s protocol. The relative expression of miRNA and its target genes was calculated by the 2–∆∆Ct method. All primers used are listed in Supplementary Table S6 .
8
Gene Expression Analysis by RT-PCR
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The gene expression was examined by performing an RT-PCR analysis using previously described protocols55 (link). Total RNA was extracted using TRIZOL reagent and reversely transcribed using a first-strand cDNA synthesis kit (Takara, China) according to the manufacturer’s protocol. Real-time RT-PCR was performed using SYBR Green II (Takara, China) and inventoried assay on CFX96 Real-Time PCR platform (Bio-Rad, USA). The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for normalization. The gene-specific primers were synthesized by Sangon Biotech (China), and their sequences are listed in Supplementary File S4 .
9
Tomato Leaf Total RNA Extraction and RT-qPCR Analysis
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A Plant Total RNA Isolation Kit (Proteinssci, Shanghai, China) was applied to extract the total RNA from tomato leaf samples. The successfully extracted RNA samples were then reverse-transcribed into cDNA using HiScript II QRT SuperMix (Yeasen, Nanjing, China). The RT–qPCR was conducted according to the manufacturer’s protocol on a Bio-Rad CFX96 real-time PCR platform. The total volume of each reaction was 20 μl. The PCR conditions used were as follows: 95°C for 5 minutes, 40 cycles at 95°C for 10 seconds, 60°C for 30 seconds, and melting curve analysis (65–95°C, increasing 0.5°C every 5 seconds). The tubulin gene (Solyc04g077020) was used as a reference gene for data normalization. The relative gene expression levels were calculated using the 2−ΔΔCT method. Three biological replicates were conducted to calculate the mean ± standard error values. All of the primer sets used in this study are listed in Supplementary Data Table S3 .
10
Quantitative Analysis of miRNA and Targets
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The expression levels of randomly selected miRNAs and their targets were examined by quantitative real time PCR (qRT-PCR). Total RNA were extracted from the four different groups of samples, with three biological replications for each. The primers for the miRNAs and target genes were designed based on the methods described elsewhere [14 (link), 36 (link)]. All the sequences of primers used for qRT-PCR in this study are listed in S1 Table . The qRT-PCR analysis was subjected to CFX96 real time PCR platform (Bio-Rad, Hercules, CA, USA). The PCR conditions were 50°C for 3 min, 95°C for 5 min, then 40 cycles of 95°C for 15 sec, 55°C for 30 sec, and 40°C for 10 min. All reactions were run in triplicate. The U6 and the 18S rRNA of Paulownia were chosen as the endogenous reference genes for miRNA and target mRNA normalization, respectively. The relative expression levels of the miRNAs and targets were calculated using the method of Livak and Schmittgen [37 (link)].
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