Total RNA was isolated from each organ using the RNAprep Pure Plant Plus Kit (DP441; TIANGEN Biotech Co. Ltd., Beijing, China) and reverse-transcribed to generate cDNAs. qPCR was performed using qPCR SYBR Green Premix (Vazyme, Nanjing, China) and CFX96 Touch Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The primer sequences for the ten CqTH genes were generated using Primer Premier 5.0 software [77 ] (Table S7 ). We used the GAPDH gene as the internal control because of its consistent expression across developmental stages in most plant organs [78 (link)]. qPCR was performed using three biological replicates and three technical replicates per sample. The relative expression of the target genes was determined using the 2-ΔΔCT method [79 (link)].
Qpcr sybr green premix
Manufactured by Vazyme
Sourced in China
QPCR SYBR Green Premix is a ready-to-use solution for quantitative real-time PCR (qPCR) analysis. It contains SYBR Green I, a fluorescent dye that binds to double-stranded DNA, and necessary reagents for efficient DNA amplification and detection.
Automatically generated - may contain errors
Lab products found in correlation
Rnaprep pure plant plus kit, by Tiangen Biotech (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Trizol reagent, by Tiangen Biotech (1 mentions)
Hiscript 2 1st strand cdna synthesis kit, by Vazyme (1 mentions)
Cfx real time pcr detection system, by Bio-Rad (1 mentions)
4 protocols using qpcr sybr green premix
1
Quantitative Gene Expression Analysis
2
Echinatin Modulates Gene Expression in Staphylococcus aureus
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Staphylococcus aureus USA300 culture at an initial OD600 of 0.3 was treated with echinatin (0–32 μg/mL) or DMSO, and the culture was incubated while shaking until an OD600 of 2.5 was reached. The bacteria were subsequently collected, and total RNA was extracted using TRIzol reagent (Tiangen, Beijing, China) according to the manufacturer’s instructions. Total RNA (1 μg) was transcribed into complementary DNA (cDNA) using the HiScript® II 1st Strand cDNA Synthesis Kit (#R211, Vazyme, Naijing, China). Gene-specific primers were designed based on the base sequences provided by NCBI (Table 1 ). qRT–PCR was performed using an ABI 7900HT Real-time PCR system in combination with qPCR SYBR Green premix (Vazyme, Naijing, China) and the following cycle parameters: 95°C for 30 s; and 40 cycles of 95°C for 5 s, 60°C for 30 s and 72°C for 30 s. The transcription levels, which were normalized to the 16S RNA, were calculated using the 2−ΔΔCT method.
3
Plant Total RNA Extraction and qPCR Analysis
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Total RNA of each sample was extracted using a plant RNA extraction kit (TIANGEN DP441), and the sequences were used for cDNA library construction. qPCR SYBR Green Premix (Vazyme, China) was used to conduct qPCR analysis in a CFX™ real-time PCR detection system (Bio-Rad, USA). The primer sequences used were designed by Primer 5.0 (Additional File 6 : Table S6). We used the Actin gene, which was stably expressed at each growth stage in almost all tissues, as the internal control [76 (link)]. The ACTIN gene was used as calibration to detect three technical repeats of the three biological repeats, and 2-ΔΔCT method was used to analyze the expression [77 (link)].
4
Transcriptional Profiling of S. aureus Genes
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To elucidate the effect of Mo-A on the transcriptional activity of pivotal genes in S. aureus, we cultured the bacterium in the presence of variable Mo-A concentrations, initiating at an OD600 of 0.3 and extending into the log phase. Centrifugation was utilized to collect the bacteria, and total RNA was subsequently isolated utilizing TRIzol reagent, which was then reverse transcribed into cDNA. Quantitative real-time PCR (qRT‒PCR) was carried out employing qPCR SYBR Green premix (Vazyme, Nanjing, China) along with the primers delineated in Supplementary Table 1 . The thermal cycling parameters encompassed an initial denaturation phase at 95 °C for 30 s followed by 40 cycles, each comprised of a denaturation step at 95 °C for 5 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. The obtained transcript levels were normalized to 16 S RNA and rplD which were evaluated through the 2-ΔΔCT method. All measurements were performed in triplicate.
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