RT-qPCR was used to analyze the expression of the candidate genes in the root tissues and leaf tissues from the two parents' seedlings. The SYBR Green Supermix kit purchased from Takara was used for PCR reactions, and the reaction conditions were as follows: pre-denaturation at 95°C for 2.5 min, followed by denaturation at 95°C for 10 s, and renaturation at 60°C for 34 s for a total of 40 cycles; at the end of the reaction, the system was kept at 95°C for 15 s, followed by lowering the temperature to 60°C for 1 min. The replicates were carried out for each experiment following the manufacturer's instructions. The forward primer sequence was 5′-GAAGCATTTCTCAGTGGCAA-3′, and the reverse primer sequence was 5′-ATGATGTCTCACCTCCTCCC-3′. β-actin was used as an internal reference, with a forward primer sequence of 5′-TCAAGAAGGCTATCAAGGAG-3′ and the reverse primer sequence of 5′-GTAACCCCATTCGTTGTCAT-3′.
Sybr green supermix kit
Manufactured by Takara Bio
Sourced in Japan, United States, China
The SYBR Green Supermix kit is a ready-to-use solution designed for real-time quantitative PCR (qPCR) applications. The kit contains SYBR Green I dye, a DNA-binding fluorescent dye, which enables the detection and quantification of target DNA sequences during the PCR amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (12 mentions)
Primescript rt reagent kit, by Takara Bio (7 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Lightcycler 96 real time pcr system, by Roche (2 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (2 mentions)
Rt reagent kit, by Takara Bio (2 mentions)
Qiazol reagent, by Thermo Fisher Scientific (2 mentions)
Abi 7900 instrument, by Thermo Fisher Scientific (1 mentions)
Exscript rt reagent kit, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Superscript first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Minibest viral rna dna extraction kit ver 5, by Takara Bio (1 mentions)
Reverse transcriptase, by Takara Bio (1 mentions)
Lightcycler 480 2 pcr platform, by Roche (1 mentions)
Oligo dt primer, by Thermo Fisher Scientific (1 mentions)
Tri rna reagent, by Favorgen Biotech (1 mentions)
M mlv reverse transcriptase, by Enzynomics (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Cdna synthesis kit, by Takara Bio (1 mentions)
29 protocols using sybr green supermix kit
1
Gene Expression Analysis in Plant Tissues
2
Quantitative RT-PCR Analysis of Hub Genes
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Total RNA was isolated from PTC cells and tissues using the TRIzol reagent (Invitrogen, USA). cDNA was synthesized using 2.0 μg of total RNA with the PrimeScript™ RT reagent kit (TaKaRa Biotechnology, Japan). The SYBR-Green Supermix kit (TaKaRa) was employed to detect the relative mRNA expression in the ABI 7900 instrument (Applied Biosystems Inc.). The relative expression levels were determined by the 2-ΔΔCt method and normalized to internal control GAPDH [24 (link), 25 (link)]. All qPCR reactions were performed in triplicate. The primers used to explore mRNA expression of ten hub genes were shown in Table 1 .
3
Quantitative Gene Expression Analysis of Lens Capsules
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Total RNA from the lens capsules of mice was extracted using the TRIzol reagent. Reverse transcription was performed with the ExScript RT Reagent Kit (Invitrogen). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed using a SYBR Green Supermix kit (Takara, Tokyo, Japan), with β-actin as an endogenous control. The transcriptions were investigated for several target genes, including FAS (forward, 5´-TTGCTGGCACTACAGAATGC-3´ and reverse, 5´-AACAGCCTCAGAGCGACAAT-3´), p62 (forward, 5´-GGCGCACTACCGCGATGAGGA-3´ and reverse, 5´-TGTTCCCGCCGGCACTCCTT-3´) and β-actin (forward, 5´-TCGTGGGCCGCCCTAGGCAC-3´ and reverse, 5´-TGGCCTTAGGGTTCAGGGGGG-3´. The relative levels of each gene expression were normalised to β-actin and calculated using the 2−△△CT method.
4
Real-time qPCR Quantification of Gene Expression
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SYBR Green Supermix Kit (Takara, Kusatsu, Japan) was used to perform a real-time quantitative PCR (RT-qPCR) on the BIO-RAD CFX96™ Real-Time System. RT-qPCR reaction was set up as follows: 95 °C for 30 s, 40 cycles at 95 °C for 5 s to denature DNA, and 60 °C for 34 s to anneal. The relative expression levels were conducted based on the 2–ΔΔCt method [34 (link)]. A stably expressed reference gene 18s rRNA was selected as an internal control. All primers used were designed on Primer3web (primer3.ut.ee) (Supplementary Materials Table S2 ).
5
Cytokine and Gene Expression Analysis in Murine Lung
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Total RNA was extracted from left lung samples using TRIzol™ Reagent (Invitrogen, Carlsbad, CA, USA) and reversely transcribed to cDNA by using the Superscript First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s instructions. A quantitative analysis of the relative mRNA expression of different cytokines (TNF-α, IL-6, IL-1β, IL-10 and TGF-β), HMGB1, TLR2 and MyD88 in murine lung tissues were determined in triplicate using SYBR Green Super Mix Kit (Takara Bio Inc., Tokyo, Japan) on a Roche LightCycler® 96 real-time PCR system (Roche Molecular Systems, Inc., USA). The relative mRNA expression was calculated with the comparative △Cq method using the formula 2−△△Cq compared to GAPGH housekeeper gene control. The primers listed in Table 2
6
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from ileum and Caco-2 cell by Trizol (Invitrogen, Carlsbad, CA, USA). The concentration and purity of total RNA were measured and approximately 1 μg RNA was reverse-transcribed into cDNA-by-cDNA Synthesis kit (TaKaRa, Otsu, Shiga, Japan). Quantitative real-time PCR was performed using SYBR Green Supermix kit (TaKaRa, Otsu, Shiga, Japan). Primers used in the study are listed in Table 1 .
7
Quantitative RT-PCR for Serum ALSV
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Total RNA of all serum samples was extracted using a RNA Extraction kit (MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0; TaKaRa, Shiga, Japan) according to manufacturer’s instructions and stored at −80 °C for use. Quantitative real-time RT-PCR (RT-qPCR) was conducted on RNA of serum samples by using a SYBR Green Supermix kit (TaKaRa) according to the manufacturer’s instructions. The primers used in the system included the forward primer (5′-GGC TAA ACA CAT CAA ACA-3′) and the reverse primer (5′-GCA TCC AGG TCA TAG TTA-3′), which targeted the ALSV NS3 gene. The cut-off cycle threshold (Ct) value was set at 35 cycles for a positive reaction, and the sample was considered positive if it had a Ct value below the cut-off. A standard curve from positive control sample was used to determine the viral RNA copy numbers of the positive samples.
8
Quantitative Analysis of DTYMK Expression
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Total RNA was extracted from HCC patient tissues and cells using TRIzol Reagent (Life Technologies, USA) and reverse transcription was performed using the PrimeScript® RT reagent Kit (Takara Bio, Japan). Quantitative RT-PCR (qRT-PCR) was performed on a CFX Connect ™ Real-Time PCR Detection System (Bio-Rad, USA) with SYBR Green Supermix kit (Takara Bio, Japan) according to the manufacture’s instruction. The primer sequences were listed as follows:
DTYMK(forward):5′-CCGGTTCCCGGAAAGATCAAC-3′;
DTYMK(reverse):5′- TCCCAGCGATTTGCAGAAAAA-3′.
DTYMK(forward):5′-CCGGTTCCCGGAAAGATCAAC-3′;
DTYMK(reverse):5′- TCCCAGCGATTTGCAGAAAAA-3′.
9
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was isolated from cultured cells using TRIzol reagent (Invitrogen) and reverse transcribed using the PrimeScript RT reagent kit (Takara) according to the manufacturer's instructions. qRT‐PCR was performed using a SYBR Green Supermix kit (Takara), with β‐actin as an endogenous control. The primer sequences used for PCR are shown in Table S1
10
Quantitative Analysis of Gene Expression in HCC
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Total RNA from 100 fresh-frozen HCC tissue samples was extracted by TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) and reverse transcribed using the PrimeScript® RT reagent Kit (Takara Bio, Dalian, China) according to the manufacturer's protocol. Further, quantitative real-time PCR was performed using the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and the SYBR Green Supermix kit (Takara Bio) according to the manufacturers' instructions. The expression levels of each genes were analyzed using the 2-ΔCT method with Homo sapiens GAPDH as the control housekeeping gene. We normalized the expression of the four genes using the scale function in R software, a generic function whose default method centered and scaled the data. The primers used in this study are summarized in Table S1 .
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