Fastsybr mixture

Manufactured by CWBIO

Sourced in China

FastSYBR mixture is a ready-to-use solution for quantitative real-time PCR (qPCR) that contains all the necessary components for the amplification and detection of DNA sequences.

Automatically generated - may contain errors

Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (6 mentions) Trizol, by Thermo Fisher Scientific (3 mentions) Ultrapure rna kit, by CWBIO (3 mentions) Abi 7500 real time pcr system, by Thermo Fisher Scientific (2 mentions) Revertaid rt reverse transcription kit, by Thermo Fisher Scientific (2 mentions) Rnase free dnase 1, by Takara Bio (2 mentions) Hifiscript cdna synthesis kit, by CWBIO (2 mentions) Steponeplus, by Thermo Fisher Scientific (2 mentions) Rnase free dnase 1 treatment, by Takara Bio (1 mentions) Trizol reagent, by Tiangen Biotech (1 mentions) Primescript rt master mix kit, by Takara Bio (1 mentions) Reverse transcription system, by Promega (1 mentions) Ex taq, by Takara Bio (1 mentions) Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions) M mlv reverse transcriptase, by Promega (1 mentions) Trnzol reagent, by Tiangen Biotech (1 mentions) Primescript reverse transcription kit, by Takara Bio (1 mentions) Cfx connect, by Bio-Rad (1 mentions) Abi 7500 fast system, by Thermo Fisher Scientific (1 mentions) Stool genomic dna kit, by CWBIO (1 mentions)

24 protocols using fastsybr mixture

Quantitative PCR Analysis of Gene and miRNA Expression

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RNA from tissues and cultured cells was extracted with Total RNA purification kit (GeneMark), and RNA from sorted cells was extracted with Trizol reagent (Invitrogen). Complementary DNA was synthesized from 1 mg total RNA by M-MLV reverse transcription (Takara). qPCR was performed using FastSYBR mixture (CWBIO) with specific primers (Table S2) on a real-time PCR system (StepOnePlus; Applied Biosystems). The comparative threshold cycle method and an internal control (Gapdh) were used to normalize the expression of target genes.
To measure mature miRNA, cDNA was prepared from total RNA with the TaqMan microRNA Reverse Transcription Kit (Applied Biosystems). qPCR was performed with TaqMan microRNA assays according to the manufacturer’s recommendations (Applied Biosystems). U6 small nuclear RNA was used as internal control to normalize the expression of miRNAs.
The abundance of specific intestinal bacterial groups was measured by qPCR with FastSYBR mixture (CWBIO) and universal 16s rDNA primers (Table S2). Bacterial abundance was determined using standard curves with reference to cloned bacterial DNA corresponding to a short segment of the 16s rRNA gene that was amplified using conserved specific primers. It should be noted that qPCR measures 16s rRNA gene copies per sample, not the actual bacterial numbers or CFUs.

Kang L., Zhang X., Ji L., Kou T., Smith S.M., Zhao B., Guo X., Pineda-Torra I., Wu L, & Hu X. (2020). The colonic macrophage transcription factor RBP-J orchestrates intestinal immunity against bacterial pathogens. The Journal of Experimental Medicine, 217(4), e20190762.

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Quantification of SCMV RNA and Maize Gene Expression

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Total RNA was isolated using TRIzol reagent (Tiangen, Beijing, China) followed by RNase‐free DNase I treatment (TaKaRa, Dalian, China). First‐strand cDNA was synthesized using 2.0 μg total RNA per 20 μL reaction and an oligo (dT) primer. Ten‐fold diluted cDNA, a set of gene‐specific primers (Table S1) and a FastSYBR mixture (CWBIO, Beijing, China) were mixed for qPCR to determine the accumulation level of SCMV RNA and maize genes on an ABI 7500 Real Time PCR system (Applied Biosystems Inc., Foster City, CA, USA). The expression level of ZmUbi mRNA was determined and used as an internal control. The relative expression level of each gene was calculated using the 2^–ΔΔCt method (Livak and Schmittgen, 2001). Differences between the treatments were then analysed using Student’s t‐test. All experiments were carried out at least three times.

Yuan W., Jiang T., Du K., Chen H., Cao Y., Xie J., Li M., Carr J.P., Wu B., Fan Z, & Zhou T. (2019). Maize phenylalanine ammonia‐lyases contribute to resistance to Sugarcane mosaic virus infection, most likely through positive regulation of salicylic acid accumulation. Molecular Plant Pathology, 20(10), 1365-1378.

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Isolation and Analysis of Intestinal Epithelial Cells

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The colons were immediately frozen in liquid nitrogen and homogenized with Trizol (Life Technologies, Carlsbad, CA, USA). Total RNA was isolated from the homogenized colons according to the manufacturer's instructions for Trizol. cDNA was synthesized from 1 μg total RNA using a RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative real-time PCR was performed using FastSYBR mixture (CWBIO) with specific primers (Supplementary Table 1). An internal control (Actb) was used to normalize the expression of target genes. For the IEC analysis, before Trizol treatment, the IECs were isolated as previously described (Becker et al., 2004 (link)) with minor modifications. Briefly, the colons were cut longitudinally and washed twice with cold PBS to remove fecal material. Subsequently, 5-mm-long pieces of colon were incubated at 37°C in PBS supplemented with 0.145 mg/ml dithiothreitol (DTT) and 0.37 mg/ml ethylenediaminetetraacetic acid (EDTA) for 15 min followed by vigorous stirring. The supernatant was passed through a 70-μm cell strainer and centrifuged at 1,000 rpm for 5 min, the pelleted IECs were collected. Total RNA was isolated from the pelleted IECs according to the manufacturer's instructions for Trizol. cDNA was synthesized as mentioned above. The cDNA was then subjected to further analysis by Quantitative real-time PCR.

Yin A., Luo Y., Chen W., He M., Deng J.H., Zhao N., Cao L, & Wang L. (2019). FAM96A Protects Mice From Dextran Sulfate Sodium (DSS)-Induced Colitis by Preventing Microbial Dysbiosis. Frontiers in Cellular and Infection Microbiology, 9, 381.

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Quantitative Real-Time PCR Protocol

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For reverse transcription of RNA extracts, complementary DNA was synthesized using PrimeScript RT Master Mix kit (Takara, RR036A). Real-time PCR was then performed using FastSYBR Mixture (CWBIO, CW0955M) according to the manufacturer's instructions.
Ct values from all total RNA samples were normalized to rp49 mRNA expression. Ct values from all nascent RNA samples were normalized to Act42A mRNA expression. Ct values from all ChIP samples were normalized to input as described in the above section (% input). All real-time PCR primer sequences are provided in Supplementary Tables S2 and S3. The MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) (45 (link)) checklist is provided in Supplementary Table S4.

Jia R., Song Z., Lin J., Li Z., Shan G, & Huang C. (2021). Gawky modulates MTF-1-mediated transcription activation and metal discrimination. Nucleic Acids Research, 49(11), 6296-6314.

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Quantitative analysis of SKP2 mRNA

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Total RNA was extracted using Trizol reagent (Invitrogen, USA). Two micrograms of RNA was reverse transcribed using the Reverse Transcription System (Promega, USA), according to the manufacturer’s recommended instructions. The cDNA was diluted ten-fold prior to PCR amplification. Real-time PCR was performed using a FastSYBR Mixture (CWBIO, China). The primer pairs that were used were as follows: SKP2, (forward) 5′-GCTGCTAAAGGTCTCTGGTGT-3′, (reverse) 5′- AGGCTTAGATTCTGCAACTTG-3′; and GAPDH, which served as an internal control, (forward) 5′-TCTCTGCTCCTCCTGTTC-3′ and (reverse) 5′-CTCCTGGAAGATGGTGATG-3′. The mRNA levels of SKP2 were quantified by measuring the threshold cycle (Ct).

Jia T., Zhang L., Duan Y., Zhang M., Wang G., Zhang J, & Zhao Z. (2014). The differential susceptibilities of MCF-7 and MDA-MB-231 cells to the cytotoxic effects of curcumin are associated with the PI3K/Akt-SKP2-Cip/Kips pathway. Cancer Cell International, 14, 126.

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Validation of Luteoloside Synthesis Genes

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To validate the accuracy of the gene expression levels of DEGs obtained from the RNA-Seq analysis, 19 genes possibly associated with luteoloside synthesis were randomly selected and subjected to qPCR detection. Gene-specific primers for the selected genes were designed using an online primer design software (https://www.genscript.com/ssl-bin/app/primer) (Supplementary Table S5) and a melting curve analysis was used to confirm specificity. qRT-PCR was performed using a Fast SYBR Mixture (CWBIO, Beijing, China) on a Bio-Rad CFX connect real-time PCR detection system using of 95 °C incubation for 10 min, then 40 cycles of 95 °C for 15 s and 60 °C for 60 s. For all qPCR experiments, three biological replicates were performed. Relative expression levels were calculated based on the 2^−ΔΔCt method using tubulin as a reference gene.

Chen Z., Liu G., Tang N, & Li Z. (2018). Transcriptome Analysis Reveals Molecular Signatures of Luteoloside Accumulation in Senescing Leaves of Lonicera macranthoides. International Journal of Molecular Sciences, 19(4), 1012.

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RBSDV S7 Gene Expression Quantification

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Total RNA was extracted from the maturing zones [23 (link)] of the first upper newly developed leaves which were harvested at 15 days post inoculation (dpi) with TRNzol reagent (Tiangen, Beijing, China) and then treated with RNase-free DNase I (TaKaRa, Dalian, China). First strand cDNA was synthesized using 1 μg total RNA, M-MLV reverse transcriptase (Promega, Madison, WI, USA) and random hexamer primer according to the manufacturer’s instructions. PCR was performed using S7-specific primers (qRP-F: 5′-GCTCCTACTGAGTTGCCTGTC-3′ and P1-R: 5′-TCAGCAAAAGGTAAAGGAAGG-3′) [24 (link)] and Ex Taq (TaKaRa, Dalian, China) according to the manufacturer’s instructions. RT-qPCR was performed using ten-fold diluted cDNA product, primers qRP-F and P1-R and FastSYBR mixture (CWBIO, Beijing, China) with ABI PRISM 7500 sequence detection system (Applied Biosystems Inc., Foster City, CA, USA). Viral concentrations were measured as described previously [19 (link),24 (link)] via comparing the threshold cycles (Ct) value of each sample with the standard curve, constructed based on Ct values of serially diluted recombinant plasmid containing a partial fragment of RBSDV S7.

Wang R., Du K., Jiang T., Di D., Fan Z, & Zhou T. (2022). Comparative Proteomic Analyses of Susceptible and Resistant Maize Inbred Lines at the Stage of Enations Forming following Infection by Rice Black-Streaked Dwarf Virus. Viruses, 14(12), 2604.

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Quantifying Ustilaginoidin Biosynthesis Genes

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Total RNAs were isolated from 7‐day‐old U. virens cultures using an Ultrapure RNA Kit (CWBIO) and cDNAs were synthesized using a PrimeScript reverse transcription kit (TaKaRa) following the manufacturers' instructions. The transcript levels of ustilaginoidin biosynthesis‐related genes were detected by RT‐qPCR. PCR primers were designed by Primer‐BLAST (https://www.ncbi.nlm.nih.gov/tools/primer‐blast/; Table S1). RT‐qPCR was performed using Fast SYBR mixture (CWBIO, Beijing). The α‐tubulin gene was used as an internal reference. The relative gene expression levels were calculated by 2^−ΔΔCt method (Schmittgen et al., 2000 (link)).

Wang B., Duan G., Liu L., Long Z., Bai X., Ou M., Wang P., Jiang D., Li D, & Sun W. (2024). UvHOS3‐mediated histone deacetylation is essential for virulence and negatively regulates ustilaginoidin biosynthesis in Ustilaginoidea virens. Molecular Plant Pathology, 25(2), e13429.

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Validating Gene Expression Levels in CGA Biosynthesis

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To validate the accuracy of the gene expression levels of DEGs obtained from the RNA-Seq analysis, six DEGs from the three tissues and five genes possibly associated with CGA synthesis were randomly selected and subjected to qPCR detection. Gene-specific primers for selected genes were designed using online primer design software (https://www.genscript.com/ssl-bin/app/primer) (S1 Table) and a melting curve analysis was used to confirm specificity. qRT-PCR were performed using a Fast SYBR Mixture (CWBIO, Beijing) on an Bio-Rad CFX connect real-time PCR detection system using of 95°C incubation for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 60 s. For all qPCR experiments, three biological replicates were performed. Relative expression levels were calculated based on the 2^-ΔΔCt method using tubulin as a reference gene.

Chen Z., Tang N., You Y., Lan J., Liu Y, & Li Z. (2015). Transcriptome Analysis Reveals the Mechanism Underlying the Production of a High Quantity of Chlorogenic Acid in Young Leaves of Lonicera macranthoides Hand.-Mazz. PLoS ONE, 10(9), e0137212.

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Quantification of PRDX2 Expression

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Total RNA was extracted from NSCLC cells transfected with siRNAs using an Ultrapure RNA Kit (CWBIO, Beijing, China) and reverse transcripted into cDNA by the HiFiScript cDNA Synthesis Kit (CWBIO). The PCR reaction was performed with FastSYBR Mixture (CWBIO) on an ABI 7500 Fast system (Applied Biosystems, Foster, CA). The following primers were used in this study: PRDX2, 5′-CCTTCAAAGAGGTGAAGCTG-3′ (forward) and 5′-GTTGCTGAACGCGATGAT-3′ (reverse); β-actin was used as the internal control, 5′-CCCGAGCCGTGTTTCCT-3′ (forward) and 5′-GTCCCAGTTGGTGACGATGC-3′ (reverse). The 2^-ΔΔCt method was used to calculate the relative expression of PRDX2.

Jing X., Du L., Niu A., Wang Y., Wang Y, & Wang C. (2020). Silencing of PRDX2 Inhibits the Proliferation and Invasion of Non-Small Cell Lung Cancer Cells. BioMed Research International, 2020, 1276328.

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