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Aceq sybr qpcr master mix

Manufactured by Vazyme
Sourced in China

AceQ SYBR qPCR Master Mix is a ready-to-use solution for real-time quantitative PCR (qPCR) reactions. It contains all the necessary components, including a hot-start DNA polymerase, buffer, and SYBR Green I dye, for efficient and sensitive DNA amplification and detection.

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15 protocols using aceq sybr qpcr master mix

1

Quantitative RNA Expression Analysis

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Total RNA from 10 × 106 bone marrow cells was isolated using RNeasy Mini Kit (QIAGEN, Hilden, Germany). DNase treatment was applied using DNA-free DNase Treatment and Removal Reagents (Ambion, Austin, TX, USA). cDNA was synthesized from 1 µg of RNA using TaqMan Reverse Transcription Reagents (Thermo Fisher Scientific, Waltham, MA, USA) and oligo(dT) primers in a final volume of 25 μL. Real-time quantitative PCR reactions were performed in technical triplicates in 20 μL volume using AceQ SYBR qPCR Master Mix (Vazyme Biotech Co., Nanjing, China) and the respective primers (Supplementary Table S1) using a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). Primer set efficiency was first tested in a standard curve experiment. Relative expression was calculated from Ct obtained from Applied Biosystems ViiA 7 Real-Time PCR System software (Thermo Fisher Scientific). Since test and housekeeping primer set efficiencies differed >10%, we applied the corrective formula described in [40 (link)] to calculate the relative expression ratio. β-actin or Hprt expression was used for normalization (Supplementary Table S1).
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2

Quantitative Analysis of Drought Stress Genes

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Quantitative real-time RT-PCR was used to analyse the expression of several drought-marker genes and the ATG8 gene family. Total RNA was extracted from Arabidopsis leaves using Qiagen RNeasy Plant Mini Kit. RNA was reverse transcribed using oligo (dT) and the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions. Gene-specific primers for each gene were designed using Vector NTI Advance 10 software (Supplementary Table S1). Real-time PCR was performed using AceQ SYBR qPCR Master Mix (Vazyme), and the signals were detected on an iCYCLER (Bio-Rad) according to the manufacturer’s instructions. The cycling profile consisted of 95 °C for 10 min followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. The expression levels of the genes of interest were normalized to the constitutive UBQ10 gene by subtracting the cycle threshold value of UBQ10 from the cycle threshold value of the gene. The results are shown as means ±SD for at least three independent RNA samples.
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3

Quantitative PCR Protocol for Gene Expression

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For the qPCR, 1.25 µL Primer (Table A1 Appendix A) (QIAGEN GmbH, Hilden, Deutschland), 5µL AceQ SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China), and 0.5 µL cDNA were pipetted into one well of a 96-well plate according to the AceQ SYBR qPCR Kit (Vazyme Biotech, Nanjing, China). Beta-glucuronidase (GUSB) and TATA-binding-protein (TBP) were used as reference genes. From the Ct value and the calculated ΔCt and ΔΔCt values, the fold change (2ΔΔCt) was calculated. The following steps were repeated 40 times: initial denaturation to 95 °C for 5 min, denaturation at 95 °C for 10 s, and primer hybridization at 60° for 30 s. Elongation occurred when the sample was being heated up for the denaturation phases. The software QuantStudio™ Design & Analysis (Thermo Fischer, Waltham, MA, USA) was used for evaluation.
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4

m6A RNA Immunoprecipitation and qPCR Analysis

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Firstly, the collected RNA was fragmented. Secondly, protein A magnetic beads (Thermo Fisher Scientific, Waltham, MA, USA) were incubated with anti-m6A antibody (abcam, Cambridge, UK) at room temperature for 1 h. Thirdly, the recovered RNA fragments were co-incubated with the mixture of magnetic beads and antibodies at 4 °C for 3 h. Fourthly, the above mixture was eluted with elution buffer, and the collected RNA was reverse-transcribed by HiScript reverse transcriptase. Lastly, the corresponding mixture of cDNA was configured according to AceQ SYBR® qPCR Master Mix (Vazyme), and the expression levels of BCL2 and TLR4 were detected using a ViiA7 Real-Time PCR System instrument. The percentage input method was used for data analysis, where % input = 2−(Average CTRIP − AverageCTinput − log2(input dilution factor)). Related primer sequences were as follows: TLR4 F, CCGGCTGGTTTTGGGAGAAT and R, ATGGTCAGGTTGCACAGTCC; BCL2 F, CAGTTGCTCTGCTGTTTGAGG and R, CATTACTCTAGTGCTCCCCGC.
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5

Comprehensive RNA Isolation and Quantification

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Total RNA in tissue specimens and cultured cells was isolated using TRIzol™ Plus RNA Purification Kit (Thermo Fisher). Isolation of cytoplasmic and nuclear RNA from NSCLC cells was performed using the Cytoplasmic & Nuclear RNA Purification Kit (Norgen, Thorold, ON, Canada) in accordance with the manufacturer’s operating procedures. For RNase R digestion, total RNA derived from NSCLC cells was digested with 3 U/mg RNase R (Thermo Fisher). The reverse transcription reaction was performed using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) or miRNA 1st Strand cDNA Synthesis Kit (Vazyme). After mixing AceQ SYBR qPCR Master Mix (Vazyme) with complementary DNA, qPCR was run on the Roche Applied Science LightCyclerTM 480 (Roche, Basel, Switzerland). Relative RNA levels of genes were calculated by the 2−ΔΔCt method using U6 or GAPDH as the reference gene for normalization. Primer sequences are listed in Table 1.
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6

RNA Extraction and qPCR Analysis

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Total RNA was collected from tumor tissues using an Animal Total RNA Isolation Kit (Foregene Co., Ltd, China) following the manufacturer’s protocol. cDNA was prepared using HiScript II Q RT SuperMix for qPCR (+gDNA Wiper) (Vazyme Biotech Co., Ltd., China). The qPCR was performed with AceQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., China) and the gene expression was normalized to the housekeeping gene ActB.
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7

Quantitative RT-PCR Analysis of CYB561 and H2AFY Expression

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Total RNA was extracted by TRIzol reagent (Ambion, USA). Nest, reversed transcription of RNA to cDNA using the PrimeScript™ RT MasterMix kit (Takara, Japan). qRT-PCR was performed using AceQ SYBR qPCR Master Mix (Vazyme, China) according to the manufacturer’s instructions. The primers sequences are used as follows: CYB561 forward primer: GTCTTCAGGAACGAAGCTAAAC, reverse primer: CTTCTTCCTGTGGTAGTCGAAC. H2AFY forward primer: TGCTGCGGTACAT CAAGAAAGGC, reverse primer: CCCCACTGGCTATGGTGACTCC.
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8

Quantitative Gene Expression Analysis

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Total RNA was extracted by RNeasy Mini Kit (QIAGEN, Hilden, Germany) from 10 × 106 bone marrow or spleen cells previously lysed in RLT buffer (QIAGEN) supplemented with 1% β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and stored at −80 °C. RNA was treated with DNA-free DNase Treatment and Removal Reagents (Ambion, Austin, TX, USA). DNase-treated total RNA (1 µg) was reverse transcribed with oligo(dT) and TaqMan Reverse Transcription Reagents (Thermo Fisher Scientific, Waltham, MA, USA). The resulting cDNA was diluted tenfold before use. Real-time quantitative PCR was performed in technical triplicates using AceQ SYBR qPCR Master Mix (Vazyme Biotech Co., Nanjing, China) and the corresponding primers (Supplementary Table 1) using a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). All RT–qPCR analyses were performed on an Applied Biosystems QuantStudio Software V1.3 (Thermo Fisher Scientific). Primer set efficiency was firstly calculated in a standard curve experiment. Relative gene expression levels were calculated using the comparative CT method. Since target and housekeeping primer set efficiencies differed >10%, we applied the corrective formula described in [21 (link)] to calculate the relative expression ratio. Gene expression levels were then normalized to that of Hprt.
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9

Real-Time PCR Analysis of Inflammatory Cytokines

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HiScript reverse transcriptase (Vazyme, Nanjing, China) was used to reverse-transcribe the RNA samples. The corresponding mixture of cDNA was configured according to AceQ SYBR® qPCR Master Mix (Vazyme), and the expression levels of IL-1β, IL-6, and TNF-α were detected using a ViiA7 Real-Time PCR System (Applied Biosystems Inc., Foster, CA, USA) instrument. The 2−ΔΔCt method was used for data analysis. Related primer sequences were as follows: β-actin F, AGATCAAGATCATCGCGCCC and R, TAACGCAGCTAACAGTCCGC; IL-1β F, TTCCATATTCCTCTTGGGGTAGA and R, AAATGAACCGAGAAGTGGTGTT; IL-6 F, CAGCAGGTCAGTGTTTGTGG and R, CTGGGTTCAATCAGGCGAT; TNF-α F, TCTTCTCAAGCCTCAAGTAACAAGC and R, CCATGAGGGCATTGGCATAC.
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10

m6A RNA Immunoprecipitation and RT-qPCR

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The following experiments were performed in accordance with the manufacturer's instructions (Millipore, Bedford, MA, USA): (1) total RNA was fragmented by Zn2+ at 94°C; (2) magnetic beads (Thermo Fisher Scientific, Waltham, MA, USA) and m6A antibody (Abcam, Cambridge, UK) were incubated for 1 h at room temperature; (3) the system was incubated with the fragmented RNA at 4°C for 2 h; (4) elution buffer was used to elute the mixture twice at 4°C for 1 h; and (5) the collected eluate was subjected to RNA extraction and reverse transcription (Vazyme). In accordance with manufacturer's the instructions, cDNA was detected by RT-qPCR using the AceQ SYBR qPCR Master Mix (Vazyme). The data were analyzed by % input; that is, %input = 2^−(Average CTRIP − Average CTinput − log2(input dilution factor)). CTRIP means the CT value of the RNA immunoprecipitation (IP RNA) samples, and CTinput means the CT value of the input RNA samples. The primers for the relevant methylated RNA were as follows (Table 2).
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