Transstart probe qpcr supermix

Manufactured by Transgene

Sourced in China, United States

TransStart Probe qPCR SuperMix is a ready-to-use solution for probe-based quantitative real-time PCR (qPCR) applications. It contains all the necessary components, including a high-performance DNA polymerase, buffer, and probes, to perform qPCR reactions efficiently.

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Lab products found in correlation

Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (2 mentions) Cfx96 real time system, by Bio-Rad (1 mentions) Easyscript one step gdna removal and cdna synthesis supermix, by Transgene (1 mentions) Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions) Nuclease free water, by Transgene (1 mentions) Mastercycler ep realplex, by Eppendorf (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Hiscript 2 rt supermix, by Vazyme (1 mentions) Sybr green master mix, by Novogene (1 mentions) Rnaiso plus, by Takara Bio (1 mentions) Cfx 96 fluorescent quantitative pcr instrument, by Bio-Rad (1 mentions) Abi prism 7700, by Thermo Fisher Scientific (1 mentions) Trizol, by Tiangen Biotech (1 mentions) Tianscript rt kit, by Tiangen Biotech (1 mentions) Primescript rt reagent kit, by Takara Bio (1 mentions) Transscript 2 first strand cdna synthesis supermix, by Transgene (1 mentions) Rna easy kit, by Transgene (1 mentions) Trizol solution, by Transgene (1 mentions) Abi steponeplus pcr system, by Thermo Fisher Scientific (1 mentions)

11 protocols using transstart probe qpcr supermix

Optimized Multiplex RT-qPCR for Coronavirus Detection

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All real-time qPCR reaction systems were set to a volume of 20 μL. For single qPCR amplifying TGEV, PEDV, and PDCoV, 10 μL 2 × TransStart Probe qPCR SuperMix (TransGene, Beijing, China), 200 nM primers and probe each, 1 μL plasmid DNA template, and 6.8 μL nuclease-free water were pooled and mixed. For PEAV amplification, 10 μL 2 × TransStart Probe qPCR SuperMix (TransGene, Beijing, China), 500 nM each primer, 100 nM probe, 1 μL plasmid DNA template, and 6.8 μL nuclease-free water were pooled and mixed. All reactions were amplified on a Bio-Rad CFX96™ Real-time System (Bio-Rad, Hercules, CA, USA) at 94 °C for 30s, followed by 40 cycles of 94 °C for 5 s and 60 °C for 30s.
For reaction system of multiplex real-time PCR, 10 μL 2 × TransStart Probe qPCR SuperMix combined with all primers, probes, templates, and nuclease-free water to a final volume of 20 μL. The concentrations of each primer and probe of PEDV, PDCoV, TGEV, and PEAV were optimized for better outputs. The amplifying cycles of multiplex qPCR were carried out as same as singular real-time qPCR. The Cq value higher than 35 was considered negative. All qPCR results were analyzed by CFX Manager™ software.

Huang X., Chen J., Yao G., Guo Q., Wang J, & Liu G. (2019). A TaqMan-probe-based multiplex real-time RT-qPCR for simultaneous detection of porcine enteric coronaviruses. Applied Microbiology and Biotechnology, 103(12), 4943-4952.

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Comprehensive Transcriptome Analysis Pipeline

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RNA extraction and reverse transcription were performed according to the instructions in the Plant RNA maxi kit (Biomiga, San Diego, CA, USA) and the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech Co., Beijing, China), respectively. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) analysis was performed using TransStart Probe qPCR SuperMix (TransGen Biotech Co.) and detected using a CFX96 Touch™ Real-Time PCR Detection System (Bio-rad Laboratories, Inc., Singapore, Singapore). Relative expression levels were calculated using the 2^−△△Ct method [50 (link)]. All RT-qPCR primers are listed in Table S1. In this study, Tubulin (accession No. AB239680.1) was used as an internal reference [4 (link)]. The transcriptome sequencing data have been deposited in the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under accession numbers SUB2967341 [6 (link)].

Cheng X., Li G., Manzoor M.A., Wang H., Abdullah M., Su X., Zhang J., Jiang T., Jin Q., Cai Y, & Lin Y. (2019). In Silico Genome-Wide Analysis of Respiratory Burst Oxidase Homolog (RBOH) Family Genes in Five Fruit-Producing Trees, and Potential Functional Analysis on Lignification of Stone Cells in Chinese White Pear. Cells, 8(6), 520.

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Optimized Real-Time qPCR Conditions

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The optimization of conditions for real-time qPCR method was carried out in triplicates using TransStart Probe qPCR SuperMix (TransGen Biotech, Beijing, China) on Mastercycler ep realplex (Eppendorf, Hamburg, Germany), based on the manufacturer's instructions. Different concentrations of the primers and probe were prepared into reaction tubes to optimize the assay by evaluating the highest fluorescence and lowest threshold cycle (C_T). The optimized reaction was carried out in a 20 μl reaction system containing 10.0 μl of supplied master mix, 0.4 μl of each primer (qPCMV-F, qPCMV-R and qPCMV-P, 10 μM each), 2 μl templates DNA, and 6.8 μl of Nuclease-free Water (TransGen Biotech, Beijing, China). The thermal profile for the real-time PCR was 94°C for 50s, followed by 40 cycles of 94°C for 5 sec, 60°C for 35 sec. The ten-fold dilution of plasmid from 5.19 × 10⁸ to 5.19 × 10¹ copies/μl was used as target DNA to generate the standard curve.

Chen R., Chen Q., Wu X., Che Y., Wang C., Wang L., Yan S, & Zhou L. (2020). Development of a TaqMan Based Real-Time Fluorescent Quantitative PCR Assay for Detection of Porcine Cytomegalovirus in Semen. BioMed Research International, 2020, 5673145.

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PEDV Genome Detection via qPCR

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Anal swabs and intestinal contents in sterile PBS were vortexed thoroughly and subjected to centrifugation at 12,000 rpm for 10 min at 4 °C. Intestines were homogenized in pre-chilled sterile PBS containing 0.5 mm silicon beads with a bead-beater (Eppendorf) and centrifuged at 12,000 rpm for 10 min at 4 °C. The total RNAs from each sample were extracted from supernatants using Trizol reagent (Invitrogen) and reverse-transcribed into cDNAs using random primers. The cDNAs were applied to a Taqman probe-based real-time qPCR analysis for detection PEDV genome using the ABI 7500 system with TransStart Probe qPCR Supermix (Transgen Biotech). The detailed information of the primer set and probe targeting PEDV M gene were as follows: forward primer: 5′-GAT ACT TTG GCC TCT TGT GT-3′, reverse primer: 5′-CAC AAC CGA ATG CTA TTG ACG-3′, and Taqman probe: 5′-FAM-TTC AGC ATC CTT ATG GCT TGC ATC-TAMRA-3'. The amplification was carried out with denaturing at 95 °C for 30s, followed by 40 cycles at 94 °C for 5s and 60 °C for 30s. The genome copies were calculated based on a standard curve.

Yang S., Li Y., Wang B., Yang N., Huang X., Chen Q., Geng S., Zhou Y., Shi H., Wang L., Brugman S., Savelkoul H, & Liu G. (2020). Acute porcine epidemic diarrhea virus infection reshapes the intestinal microbiota. Virology, 548, 200-212.

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Quantification of Hepatitis B Biomarkers

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Serum and intrahepatic HBsAg titers were measured using an HBsAg chemiluminescent quantitation kit purchased from Wantai Biological Pharmacy Enterprise Co., Ltd. HBV‐DNA in serum was extracted using the Universal Genomic DNA Extraction Kit (GenMagBio, CN). Real‐time PCR was conducted to measure the level of HBV‐DNA in the serum using TransStart Probe qPCR SuperMix (TransGen Biotech, CN).

Jiang Y., Chen X., Ye X., Wen C., Xu T., Yu C., Ning W., Wang G., Xiang X., Liu X., Wang Y., Chen Y., Liu X., Shi C., Liu C., Yuan Q., Chen Y., Zhang T., Luo W, & Xia N. (2024). A Dual‐domain Engineered Antibody for Efficient HBV Suppression and Immune Responses Restoration. Advanced Science, 11(15), 2305316.

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Quantifying Host Response to PDCoV

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To examine the host response to PDCoV infection, RNA was extracted from different tissues using RNAiso Plus (Takara, Japan) and then reverse transcribed into cDNA using HiScript II RT SuperMix (Vazyme, China). Real-time PCR analysis was employed for detection with SYBR Green Master Mix (Novogene, China) in a Bio-Rad CFX96 system. The reaction mixtures were incubated at 94°C for 30 s, followed by 40 cycles at 94°C for 5 s and 60°C for 30 s. GAPDH served as an internal control for normalization. The relative expression levels of target genes were calculated by the 2^−ΔΔCt method. The viral load was measured using TransStart Probe qPCR SuperMix (Transgen, China). Briefly, the mixtures were incubated at 94°C for 30 s, followed by 40 cycles at 94°C for 5 s and 60°C for 30 s. All primers and probes are listed in Table 3.

Wang Z., Li S., Shao Y., Lu Y., Tan C., Cui Y., Ding G., Fu Y., Liu G., Chen J, & Hu Y. (2022). Genomic characterization and pathogenicity analysis of a porcine deltacoronavirus strain isolated in western China. Archives of Virology, 167(11), 2249-2262.

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Real-Time PCR Genotyping Assay

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Primer sequences and their Taqman probe sequences at SNP site (Table II) designed by Oligo6.0 were used. Primer synthesis was accomplished by Sangon Biotech Co., Ltd. (Shanghai, China). DNA solution (1 µl) and 1.2 µl prepared primer solution (including 0.4 µl upstream primer, 0.4 µl downstream primer and 0.4 µl probe primer) were added to 17.8 µl pre-prepared TransStart Probe qPCR SuperMix (Beijing TransGen Biotech Co., Ltd., Beijing, China), slightly shaken to mix, and placed into a CFX96 fluorescent quantitative PCR instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Reaction conditions: i) 94°C for 3 min, for 1 cycle; ii) 94°C for 15 sec and 60°C for 60 sec, for 42 cycles. After each cycle, the fluorescence value was read once. The experimental results were generated by built-in software of the instrument. Three replicate wells were made for each sample, diethyl pyrocarbonate (DEPC) water was used as negative control, and positive plasmid containing the sequence (synthesized by Sangon Biotech) was used as positive control. Determination of genotypes: The wild homozygous genotype was near the FAM abscissa, the mutant homozygous genotype was near the VIC ordinate, and the heterozygous genotype was near the 45° line.

Wang Z., Li Y., Wang Y., Wang X., Zhang J, & Tian J. (2018). Association between GDF5 single nucleotide polymorphism rs143383 and lumbar disc degeneration. Experimental and Therapeutic Medicine, 16(3), 1900-1904.

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Quantifying Methanol-Induced Gene Expression

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Yeast expression cultures were induced for 1 day with 0.5% methanol, and adjusted to an OD₆₀₀ of 8.0. Cell pellets from 1-mL samples of the cultures were collected by centrifugation and lysed by grinding in liquid nitrogen. Total RNA was isolated using TRIzol (TIANGEN, Beijing, China) following the manufacturer’s instructions, and cDNA was synthesized using the TIANScript RT Kit (TIANGEN, Beijing, China). Duplicate PCR reactions were performed on an ABI PRISM 7700 sequence detection system (Applied Biosystems, Weiterstadt, Germany) with standard conditions (50°C for 2 min; 95°C for 10 min; and 45 cycles of 95°C for 15 sec, and 60°C for 1 min) using TransStart Probe qPCR SuperMix (Transgen, Beijing, China) following the manufacturer’s recommendations. The primers used for mph were mph-mF and mph-mR (Table S1 in File S1). Yeast gapdh was amplified as a control for normalization using the primers GAPDH-F and GAPDH-R (Table S1 in File S1). All primers were used at a final concentration of 0.2 µM.

Wang P., Huang L., Jiang H., Tian J., Chu X, & Wu N. (2014). Improving the Secretion of a Methyl Parathion Hydrolase in Pichia pastoris by Modifying Its N-Terminal Sequence. PLoS ONE, 9(5), e96974.

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Quantitative PCR Plasmid DNA Quantification

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The plasmid DNA was amplified using the primers and probe by qPCR. qPCR was carried out using the QuantStudio 6 Flex Real-time PCR System (Thermo Fisher Scientific, USA) and TransStart Probe qPCR SuperMix (TransGen Biotech Co., Ltd., Beijing, China). The standard curve was generated by using tenfold serial dilutions of standard plasmid DNA. The cycle threshold (Ct) value of the sample was used to calculate the copy number of each sample. The 25 μL qPCR reaction comprised 10 μL of 2 × TransStart Probe qPCR SuperMix, 5 μL of plasmid DNA, 1 μL of each of the primers, 0.8 μL of probe, and RNase-free H₂O to reach the final volume. The reaction conditions for qPCR were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 58 °C for 45 s.

Yu Z., Zhao Z., Chen L., Yan H., Cui Q., Ju X., Yong Y., Liu X., Ma X, & Zhang G. (2022). Development of a droplet digital PCR assay to detect bovine alphaherpesvirus 1 in bovine semen. BMC Veterinary Research, 18, 125.

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Quantitative Real-Time PCR Analysis

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Total RNA was extracted with Trizol reagent (Meilunbio, Dalian, China) and reverse transcription using PrimeScript® RT reagent kit (Takara, Dalian, China). Quantitative real-time PCR detection of cDNA was performed using TransStart Probe qPCRSuperMix (Transgen, Beijing, China) with a thermal profile of 94 °C for 5 min followed by 40 cycles of 94 °C for 5 s, 60 °C for 30 s. The primers for RT-qPCR were as follows: GAPDH forward, 5′-AGAAGGCTGGGGCTCATTTG-3′, GAPDH reverse, 5′-AGGGGCCATCCACAGTC TTC-3′; RRS1 forward, 5′-CCCTACCGGACACCAGAGTAA-3′, RRS1 reverse, 5′-CCGAAAAGGGGTTGAAACTTCC-3′; NFKBIE forward, 5′-TTACCCATGTTGGGT CAGCC-3′, NFKBIE reverse, 5′-AACGGTGTTTCAGGGTCCTC-3′; OTUD7B forward, 5′-AATGCCCATCTGTGCCTTCC-3′, OTUD7B reverse, 5′-GTTCAAACG CCCTGCCTGTT-3′; TNF forward, 5′-AGCTGGAGAAGGGTGACCGA-3′, TNF reverse, 5′-CACAGGGCAATGATCCCAAAG-3′; NOD1 forward, 5′-TGGAGA CACCGGAGTATGTG-3′, NOD1 reverse, 5′-GAGGAGGGTAGCCTAGCAGA-3′; PI3K forward, 5′-CGAGAGTGTCGTCACAGTGTC-3′, PI3K reverse, 5′-TGTTCG CTTCCACAAACACAG-3′; Akt forward, 5′-CCTCCACG ACATCGCACTG-3′, Akt reverse, 5′-TCACAAAGAGCCCTCCATTATCA-3′. PCR reactions were carried out in triplicate (Roche lightcycle96 real-time fluorescence quantitative PCR System), and the relative amount of RRS1 expression was normalized to GAPDH by using the 2 -△△CT method.

Zhang X., Liu C., Cao Y., Liu L., Sun F, & Hou L. (2022). RRS1 knockdown inhibits the proliferation of neuroblastoma cell via PI3K/Akt/NF-κB pathway. Pediatric research.

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