For gene expression of HSP70, HSP90, HSF1, HSF3, SOD, and CAT, specific primers (Table 2 ) were used to amplify gene products. In this regard, a real-time PCR (qPCR) was performed using the SensiFAST SYBR Lo-Rox kit (Bioline, United Kingdom) and PikoReal™ 24 Real-Time PCR System (PikoReal 24, Thermoscientific, TCR0024). The reaction mix consisted of 10 μl of SensiFAST SYBR Lo-Rox mastermix, 0.5 μM of each prime, and 2 μl of cDNA. The thermal cycling conditions were initial denaturation at 95°C for 15 min, followed by 40 cycles at 95°C for 15 s, and annealing for 1 min at 60°C for all genes. Dissociation curve analyses were performed beginning at 65°C and ending at 95°C, with incremental increases of 0.5°C every 5 s to validate the specificity of the PCR products. For all tested genes, dissociation curve analysis showed only one peak at the specific melting temperature (data not shown), showing that the PCR products were specifically amplified. All genes were tested in duplicates for three birds of each chicken strain. CT values for each sample were determined and incorporated in “fold change” calculation based on the Livac method [34 (link)], and mRNA expressions for each sample were normalized against β-actin and GAPDH.
Pikoreal 24 real time pcr system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PikoReal™ 24 Real-Time PCR System is a compact, high-performance thermal cycler designed for real-time PCR analysis. It features 24 reaction wells and can accommodate a variety of sample volumes and reaction tube formats. The system provides accurate temperature control and precise detection of fluorescent signals for quantitative gene expression analysis and other real-time PCR applications.
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Lab products found in correlation
Sensifast sybr lo rox kit, by Meridian Bioscience (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rnaiso plus kit, by Takara Bio (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Pikoreal software 2, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Easytaq dna polymerase, by Transgene (1 mentions)
One step primescript rt pcr kit, by Takara Bio (1 mentions)
C1000 thermal cycler pcr, by Bio-Rad (1 mentions)
Easytaq buffer, by Transgene (1 mentions)
Trizol rna reagent, by Thermo Fisher Scientific (1 mentions)
Maxima sybr green rox qpcr master mix, by Thermo Fisher Scientific (1 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
5 protocols using pikoreal 24 real time pcr system
1
Gene Expression Analysis of Stress Markers
2
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was isolated from cells using the RNAiso Plus Kit (Takara Bio, Inc.) and cDNA was synthesized using the PrimeScript™ RT Reagent Kit (Takara Bio, Inc.) according to the manufacturers' instructions. Gene expression was detected using SYBR Premix Ex Taq™ II (Takara Bio, Inc.) according to the manufacturers' protocol on a Piko-Real 24 Real-Time PCR system (Thermo Fisher Scientific, Inc.). The primers for FOXP3, VEGF and GAPDH were synthesized by Sangon Biotech Co., Ltd. The sequences of the primers are shown in Table I . The reaction were performed as follows: 95˚C for 15 min and 40 cycles of 95˚C for 10 sec and 60˚C for 10 sec. The relative gene expression was normalized to endogenous control and expressed as 2-ΔΔCq (27 (link)).
3
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA from samples collected at 48 h (control) and 96 h (48 h after TDS) was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany). The RevertAid kit (Fermentas, ThermoFisher, Waltham, MA, USA) was used to obtain cDNA. For PCR detection, we used SYBR Green PCR Master Mix (Applied Biosystems, USA). Reactions of 12 μL were evaluated, containing 6 μL of SYBR Green PCR Master Mix (Applied Biosystems, CA, USA), 150 nM primers, and 50 ng of template cDNA. The reaction was incubated at 50°C for 2 min, 95°C for 10 min, and for 40 cycles at 95°C for 15 s and 60°C for 1 min. Amplification was carried out in a PikoReal 24 Real-Time PCR System and analyzed with PikoReal software 2.2 (ThermoFisher, Waltham, MA, USA). All samples were run in triplicate, and negative controls were included. Data was analyzed using the 2-∆∆Cq method [31 (link)] and normalized using Gst1 as an endogenous control. The primers used are listed in Table 1 .
4
Optimized DNA and RNA Amplification Protocols
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For PCR reaction, it was performed in 50 μL condition containing 5 U EasyTaq® DNA Polymerase, 5 μL 10 × EasyTaq® buffer (TransGen Biotech, Beijing, China), 0.2 mM deoxyribonucleoside triphosphates (dNTP), and 0.4 μM each of forward and reverse primer. The PCR reaction was incubated in a C1000TM Thermal Cycler PCR (Bio-Rad Laboratories, USA) for a total of 30 cycles (94 °C for 1 min, followed by 30 cycles of 15 s at 94 °C, 10 s at 55 °C and 20 s at 72 °C. A final elongation step at 72 °C for 5 min). For RT-PCR, the One Step PrimeScript RT-PCR kit (TaKaRa Bio Inc, Japan) was used. The reaction was carried out in 25 μL condition according to the manufacturer's instructions with the exception that the concentration of forward and reverse primers was 0.64 μM and the 0.4 × SYBR Green I was used to monitor the real-time amplification. The RT-PCR reaction was incubated in PikoReal 24 RealTime PCR System (Thermo Fisher Scientific Corporation, USA) at 42 °C for 5 min, 95 °C for 10 s and then 50 cycles of 5 s at 95 °C, 30 s at 55 °C followed by a signal collection.
5
Transcriptional Analysis of Infection Response in Capsicum chinense
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Cultures of C. chinense previously treated with the consortium according to the infection conditions were used to determine the transcription levels of candidate genes involved in the infection response. For the expression analysis, RNA was isolated using a TRIZol™ RNA Reagent (Invitrogen™), and the cDNA was synthesized using 500 ng of total RNA with Revert Aid Reverse Transcriptase (Thermo Scientific). Treatments at 0.5, 1, 3, 6 and 12 h with or without 1 × 10 4 cs of the consortium were analyzed; the treatment at 0.5 h was used as the control. For the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays, amplification was conducted using a Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific) and a PikoReal 24 real-time PCR system (Thermo Fisher Scientific, Ratatsie 2, FI-01620 Vantaa, Finland). The conditions of the RT-qPCR were as follows: 1) Initial denaturation step at 95 °C for 10 min; 2) Three-step cycling at 95 °C for 40 s and at Tm °C for 40 s with 40 or 45 cycles, respectively, for each gene; and 3) Final melting curve step from 56 °C to 95 °C. The primers used were designed based on pepper genome sequences (C. anuum cv. CM334 genome CDS) and tested in C. chinense (Table S1). Finally, for the fold change determination, a 2 -ΔΔ CT method and EF1α as an internal control were used.
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