SiHa cells were exposed to 0.5 × 108 cells/mL or 1.0 × 108 cells/mL CLD for 48 h. Total RNA was extracted with Trizol RNA isolation reagents (TransGen Biotech; Beijing, China), followed by cDNA synthesis with a cDNA Reverse Transcription Kit (TransGen Biotech; Beijing, China). The qPCR was conducted with a 10 μL reaction system containing 10 ng of cDNA, 5 μL of SYBR qPCR Mix (TransGen Biotech; Beijing, China), 0.4 μL of primers (10 μM), 0.2 μL of ROX, and ddH2O. A Mx3005P qPCR instrument (Agilent; Santa Clara, California) was used for detection, and data were analyzed using Mxpro software (Agilent; Santa Clara, California). Gene expression levels were normalized to β-Actin. Relevant primer sequences and reaction conditions are shown in Table S1 .
Sybr qpcr mix
Manufactured by Transgene
Sourced in China
SYBR qPCR Mix is a ready-to-use solution for quantitative real-time PCR (qPCR) analysis. It contains all the necessary components, including a DNA polymerase, buffer, and SYBR Green dye, to perform qPCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Cdna reverse transcription kit, by Transgene (1 mentions)
Mxpro software, by Agilent Technologies (1 mentions)
Mx3005p qpcr instrument, by Agilent Technologies (1 mentions)
Rna extraction kit, by Transgene (1 mentions)
M mlv reverse transcriptase, by Transgene (1 mentions)
Qtower thermal cycler, by Analytik Jena (1 mentions)
Hiscript q rt supermix for qpcr, by Vazyme (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Bio-Rad (1 mentions)
4 protocols using sybr qpcr mix
1
qPCR Analysis of CLD-Treated SiHa Cells
2
Wheat Transcriptome Analysis via RT-qPCR
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Total RNA was extracted using an RNA extraction kit (TransGen Biotech, Beijing, China) , following the manufacturer's instructions. First-strand cDNA was synthesized using oligo(dT) primers and M-MLV reverse transcriptase (TransGen Biotech). The quantitative real-time PCR was performed using SYBR qPCR Mix (TransGen Biotech) in a qTOWER thermal cycler (Analytik-Jena, Jena, Germany). All quantitative real-time PCR reactions were performed in triplicate. Wheat cyclophilin A (GenBank: AY456123.1) was used as the internal control. The primer sequences used in this work are listed in Supplemental Table 1.
3
RT-qPCR Analysis of Gene Expression
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Total RNAs were obtained from the previous samples used for sequencing. The cDNA was synthesized with the HiScript®Q RT SuperMix for qPCR (Vazyme). RT-qPCR was performed using SYBR qPCR Mix (TransGen) on the LightCycler480 System (Roche). The relative expressions of all tested candidate genes were normalized to the internal control ACTIN. Three biological replicates were performed for each RT-qPCR experiment. The relative gene expression level was calculated by 2−∆∆CT method. RT-qPCR primers were designed with Primer Premier 5.0 and the primer sequence used in this experiment is listed in Table S7 .
4
Quantitative Gene Expression Analysis
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Adherent cells were used in the logarithmic growth phase, which corresponded to a cell density of approximately 80%. TRIzol reagent (Invitrogen, CA, USA) was used to extract total RNA from the cells and transcription reagent (Transgen, Beijing, China) was then used to transcribe the RNA into cDNA. RT-PCR was performed in the 7500 Real-Time PCR System (BIO-RAD) using SYBR qPCR mix (Transgen, Beijing, China) and GAPDH as the endogenous reference [36] . The primer sequences used were: GAPDH, 5 ′ -GTCTCCTCTGACTTCAACAGCG-3 ′ (forward) and 5 ′ -ACCACCCTGTTGCTGTAGCCAA-3 ′ (reverse); GALNT3, 5 ′ -GCTGCAGTTTCATGTTAGGG-3 ′ (forward) and 5 ′ -ACAGCCCCAATAACCGTATGAA-3 ′ (reverse); OAS1, 5 ′ -GCTGGCTGAAAGCAACAGTG-3 ′ (forward) and 5 ′ -TCCAGTCCTCTTCTGCCTGT-3 ′ (reverse). The relative initial copy number of the target gene was calculated using the 2 -△△CT method.
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