Extracted RNA was used as the template for synthesizing cDNA. The total system was 20 μL, containing RNA, buffer 2× mix, and reverse transcriptase. In vitro reverse transcription was performed with incubation at 65ºC for 5 min, followed by 37ºC for 15 min and 98ºC denaturing (5 min). A real-time PCR system containing cDNA, specific primers (sequence as Table 1 ), SYBR Green mixture (Toyobo, Japan), and sterilized water, was performed in a PCR cycler (Model VIIA7, ABI, USA). Reaction conditions were: 95ºC for 5 min, followed by 40 cycles of 95ºC denature for 15 s, 60ºC annealing for 45 s, and 72ºC elongation for 15 s. Relative DNA level was determined by 2−ΔΔCt method.
Sybr green mixture
Manufactured by Toyobo
Sourced in Japan, United States
SYBR green mixture is a fluorescent dye used in molecular biology applications for the detection and quantification of DNA. It is a non-specific dye that binds to double-stranded DNA, emitting a green fluorescent signal when excited by a light source. The SYBR green mixture can be used in various DNA analysis techniques, such as real-time PCR and DNA gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Model viia7, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Cfx manager, by Bio-Rad (1 mentions)
Nanodrop 8000, by Thermo Fisher Scientific (1 mentions)
Easypure plant rna kit, by Transgene (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
High capacity cdna reverse transcription kit, by Tiangen Biotech (1 mentions)
Sybr green, by Toyobo (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rt reagent, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
7 protocols using sybr green mixture
1
Quantitative Real-Time PCR Analysis
2
Transcriptional Profiling of Walnut Flower Development
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The female flower buds were collected before, during, and after flower transition (F_1, F_2 and F_3), and leaf buds were collected during the floral transition period. The Leaf buds and female flower buds (F_1, F_2 and F_3) were collected and immediately frozen in liquid nitrogen. Total RNA was extracted with RNAout 1.0 (Tianenze, China) as described by the manufacturer and cDNA was reversed reverse-transcribed using the PrimeScript RT Reagent Kit (Takara, China). The real-time PCR analysis was performed using CFX Manager (Bio-Rad, USA) with SYBR Green mixture (Toyobo, Japan), and the walnut actin gene and walnut gadph gene were used for normalization, the amplification was applied using the cycling parameter as described previously45 (link). The results were evaluated by the 2−ΔCt method according to Livak and Schmittgen59 (link).
3
Quantitative Real-Time PCR Gene Expression
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Total RNA was extracted with an EasyPure Plant RNA Kit (Transgene, Beijing, China). RNA quantity and quality were assessed using a NanoDrop8000 (Thermo Scientific, Waltham, MA, USA). Total RNA isolation and reverse transcription with oligo (dT)18 were performed as described previously [51 (link),52 (link)]. The amounts of individual genes were quantified with gene-specific primers by real-time PCR analysis with a LightCycler 480 System (Roche, Rotkreuz, Switzerland) and SYBR Green mixture (Toyobo, Shanghai, Japan). The relative expression of specific genes was quantitated with the 2−∆∆Ct calculation method, where ΔΔCt is the difference in the threshold cycles and the reference housekeeping gene [53 (link)]. Lettuce eIF4A was used as the reference gene for normalizations, and the sequences of specific primers are shown in Table S1 .
4
Quantitative RT-PCR for Gene Expression
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Total RNA was prepared from cultured cells or mouse tissues using Trizol reagents (Invitrogen). Equal amounts of RNA were reverse transcribed with a high capacity cDNA reverse transcription kit (Tiangen) and subjected to Q-PCR by using SYBR green mixture (TOYOBO) with ABI 7900 Q-PCR Systems according to the manufacturer's protocol. All reactions were performed in triplicate, and the relative amounts of mRNAs were calculated using the comparative CT method. Primer sequences are listed in Supplementary Table 1 .
5
Validating Gene Expression via qRT-PCR
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For the verification of gene expression, cDNA were performed for qRT-PCR with a 10 µL reaction mixture containing 1 µL of cDNA, 1 µL of each reverse and forward primer, 3 µL of RNAse free water and 5 µL of SYBR green mixture (SYBR green, Toyobo, New York, NY, USA). The cDNA was synthesized using the HiScript II Q Select RT Supermix for qPCR (+gDNA wiper) cDNA synthesis kit according to the manufacturer instructions (Vazyme Biotech, Nanjing, China). qRT-PCR was performed by using a Real time PCR cycler (Eppendorf AG 22331, Hamburg, Germany) with the thermal cycling conditions as follows: 50 ℃ for 2 min, 95 ℃ for 2 min, followed by 40 cycles of 95 ℃ for 15 s, and 60 ℃ for 60 s. The cycle threshold (Ct) values were automatically calculated. The relative gene expression level was quantified using the 2−ΔΔCt method and housekeeping gene EF1-α of F. graminearum was used as the reference [34 (link)]. All primers were designed using the online NCBI primers tool software (https://www.ncbi.nlm.nih.gov/tools/primer-blast , accessed on 20 May 2021) and listed in Table S1 .
6
RNA Extraction and qRT-PCR Analysis
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Total RNAs were extracted with TRIzol reagent (Invitrogen), and 1 μg of total RNA was used for reverse transcription reaction with SuperScript III reverse transcriptase (Invitrogen). Real‐time quantitative reverse transcription PCR (qRT–PCR) analyses were performed using SYBR green mixture (TOYOBO) as described previously (Xiao et al, 2014 ), and UBIQUITIN was used as internal control. Three independent biological replicates were performed for each qRT–PCR analysis. The primers used for qRT–PCR are listed in Appendix Table S3 .
7
RNA Extraction and Quantitative PCR
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The total cellular RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s protocol, and the quantity and quality were measured by spectrophotometry. The reverse transcription (RT) reactions were performed with 1 μg of RNA using the RT reagents (Takara, Dalian, China) to synthesize cDNA according to the manufacturer’s instructions. Quantitative PCR was performed according to the manufacturer’s instructions using a SYBR green mixture (TOYOBO, Osaka, Japan).
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