Superreal premix plus sybr green kit

Manufactured by Tiangen Biotech

Sourced in China, United States, Germany, Japan

The SuperReal PreMix Plus (SYBR Green) kit is a real-time PCR reagent designed for gene expression analysis and quantification. It includes a pre-mixed solution containing SYBR Green I dye, Taq DNA polymerase, and necessary buffers and reagents for efficient real-time PCR amplification and detection.

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111 protocols using superreal premix plus sybr green kit

Quantifying Bacterial Transcriptional Response

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The primer sequence can be found in Table S1. The bacteria were pelleted when the OD₆₀₀ reached 0.6 (Table S1), and the total RNA was extracted using a total RNA isolation kit (Sangon Biotech). The RNA concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher) before cDNA synthesis using a FastKing RT Kit (Tiangen Biotech). RT-qPCR was performed using a SuperReal Premix Plus (SYBR green) Kit (Tiangen Biotech). We calculated the relative fold changes using 2^−(ΔΔCt), with 16S rRNA as the reference. All the reactions were conducted with three biological repeats.
For the antisense RNA RT-qPCR, the specific forward primers were designed to do reverse transcription using a FastKing RT Kit (Tiangen Biotech). Coupling with the corresponding reverse primer, RT-qPCR was performed using the SuperReal Premix Plus (SYBR green) Kit (Tiangen Biotech). We calculated the relative fold changes using 2^−(ΔΔCt) with 16S rRNA as the reference. All of the reactions were conducted in three biological repeats.

Hua C., Huang J., Wang T., Sun Y., Liu J., Huang L, & Deng X. (2022). Bacterial Transcription Factors Bind to Coding Regions and Regulate Internal Cryptic Promoters. mBio, 13(5), e01643-22.

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Quantitative Real-Time PCR Profiling

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Total RNA of the kidney was extracted using Trizol reagent (Tiangen, China) according to the manufacturer's protocol. The concentration and purity of isolated RNA were determined by BioSpec-nan (Shimadzu, Japan). Total RNA (1 μg) was reversely transcribed into cDNA using FastKing Reverse Transcriptase Kit (Tiangen, China). cDNA samples were used to amplify the target genes using SuperReal PreMix Plus (SYBR Green) kits (Tiangen, China) on an Eppendorf AG 22331 Real-Time PCR system (Eppendorf, Germany). Specific primers (Table 1) were synthesized by Comate, China. GAPDH expression in each sample was included as the internal control. Gene expression was quantitated using the 2^−∆∆Ct method.

Huang J., Ma X.T., Xu D.D., Yao B.J., Zhao D.Q., Leng X.Y, & Liu J. (2021). Xianling Gubao Capsule Prevents Cadmium-Induced Kidney Injury. BioMed Research International, 2021, 3931750.

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RNA Extraction and RT-qPCR Analysis

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Total RNAs were extracted from cancer samples and cultured cells with TRIzol (TaKaRa, Tokyo, Japan). mRNA was reverse-transcribed with a PrimeScript RT Reagent Kit (TaKaRa, Japan) to cDNA. GAPDH was used as the normalization control for FXYD6 and ATP-α1. Primer pair sequences are listed in Table 2. cDNA was mixed with SuperReal PreMix Plus (SYBR Green) kits (Tiangen, Beijing, China) and each primer pair to produce a 20 μL reaction mixture. All RT-PCR tests were conducted with an ABI 7500 RT-PCR system (Applied Biosystems, Carlsbad, CA, USA). The thermocycling conditions were as follows: initial denaturation at 95°C for 15 min and 45 cycles of 95°C for 10 sec, 60°C for 20 sec, and 72°C for 60 sec. Each reaction was done in triplicate, and the average 2^-ΔΔCt was used in data analysis.

Luo W., Liu Q., Chen X., Liu H., Quan B., Lu J., Zhang K, & Wang X. (2021). FXYD6 Regulates Chemosensitivity by Mediating the Expression of Na+/K+-ATPase α1 and Affecting Cell Autophagy and Apoptosis in Colorectal Cancer. BioMed Research International, 2021, 9986376.

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Quantitative Real-Time PCR Analysis of Gene Expression

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T24 and EJ-1 cells were seeded in a T25 culture flask at a seeding density of 5 × 10 5 cells/well, as mentioned in a previous study (28) (link). After adherence to the well, T24 cells were treated with 65.9 µmol L -1 MED for 48 h, and EJ-1 cells were treated with 64.6 µmol L -1 MED for 48 h. The total RNA of T24 cells and EJ-1 cells was extracted using RNAprep pure Cell/Bacteria Kit (DP430, TIANGEN Biotech Co., Ltd., China), and then reverse transcribed into cDNA with FastKing RT Kit (KR116, TIANGEN Biotech Co., Ltd.). qRT-PCR was performed using the SuperReal PreMix Plus (SYBR Green) kit (FP205, TIANGEN Biotech Co., Ltd.) to quantify the relative expression of genes. Finally, quantitative realtime PCR was performed by QuantStudio1 Real-Time PCR System (A40425, Thermo Fisher Scientific). The relative mRNA levels of each gene of interest were calculated using the 2^-ΔΔCt method. The primers were purchased from Tsingke Biotechnology Co., Ltd. ( China), and the primer sequences are shown in Table I.
Specifically, the complete qRT-PCR cycling conditions are shown in Table II.

Chen Y., Yin L., Hao M., Xu W., Gao J., Sun Y., Wang Q., Chen S., Liang Y., Guo R., Zhang J., Li J., Zhai Q., Cheng R., Wang J., Wang H, & Yang Z. (2023). Medicarpin induces G1 arrest and mitochondria-mediated intrinsic apoptotic pathway in bladder cancer cells. Acta pharmaceutica (Zagreb, Croatia), 73(2).

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Verification of miRNA-target interaction

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A total of 12 differential expressed miRNA and 11 targeted genes were selected to verify the interaction between miRNA and targets by qRT-PCR. For miRNA, forward primers were developed according to the sequence of miRNA itself (S1 Table), and universal primers provided by miRcute Plus miRNA First-Strand cDNA Kit (Tiangen, Beijing, China) were employed as reverse primers. U6 snRNA was adopted as an inner reference. The qRT-PCR were performed via miRcute Plus miRNA qPCR Kit (SYBR Green) (Tiangen, Beijing, China). The primers for target genes were designed using primer 5.0 (Primer-E Ltd., Plymouth, UK) (S2 Table). Plactin [6 (link), 49 (link)] (GenBank: JN105299; Available online: http://www.ncbi.nlm.nih.gov) was utilized as an internal control. Reverse transcription reactions and RT-qPCR reactions were completed with the FastKing RT Kit (with gDNase) (Tiangen, Beijing, China) and SuperReal PreMix Plus (SYBR Green) Kit (Tiangen, Beijing, China). All reactions were performed for three times for each sample. The comparative expressing level of miRNA and mRNA was calculated according to the 2^-ΔCt approach [50 (link)].

Xu P., Li Q., Liang W., Hu Y., Chen R., Lou K., Zhan L., Wu X, & Pu J. (2023). A tissue-specific profile of miRNAs and their targets related to paeoniaflorin and monoterpenoids biosynthesis in Paeonia lactiflora Pall. by transcriptome, small RNAs and degradome sequencing. PLOS ONE, 18(1), e0279992.

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Gene Expression Analysis in Tissues

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Brain, lung, and ileum tissues were homogenized in RNA lysis buffer. Total RNA was extracted using the Eastep total RNA extraction kit (Promega, Beijing). Equal amounts of RNA were reversely transcribed into cDNA with FastQuant RT kit (with gDNase) (Tiangen, Beijing) in accordance with the manufacturer’s instructions. Relative gene expression was determined using specific quantitative primers (see Table 1). Polymerase chain reactions were performed on a CFX96 touch system (Bio-Rad, United States) with SuperReal PreMix Plus (SYBR Green) kit (Tiangen, Beijing). The mRNA expression levels were normalized to GAPDH using the ^ΔΔCt relative to control groups.

Miao Y., Wang B., Hu J., Zhang H., Li X., Huang Y., Zhuang P, & Zhang Y. (2022). Herb Formula (GCis) Prevents Pulmonary Infection Secondary to Intracerebral Hemorrhage by Enhancing Peripheral Immunity and Intestinal Mucosal Immune Barrier. Frontiers in Pharmacology, 13, 888684.

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Quantitative Analysis of mRNA Expression

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Total RNA was isolated from tissues frozen-sectioned into thin slices and cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. cDNA was synthesized with a FastQuant RT Kit (Tiangen, China). The mRNA expression level was assessed by qRT-PCR with a SuperReal PreMix Plus (SYBR Green) Kit (Tiangen) using a LightCycler 96 (Roche, USA). All mRNA levels were normalized to the levels of GAPDH or β-actin. The primers used in qRT-PCR are listed in Supplementary Table 3.

Wu Y., Liu Q., Xie Y., Zhu J., Zhang S., Ge Y., Guo J., Luo N., Huang W., Xu R., Liu S, & Cheng Z. (2023). MUC16 stimulates neutrophils to an inflammatory and immunosuppressive phenotype in ovarian cancer. Journal of Ovarian Research, 16, 181.

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Transcriptional Analysis of FLS Resistance in Soybean

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Among the association panel, ‘HF55’ (resistant to race 1, carried all of beneficial alleles of SNPs associated FLS resistance) and ‘TD50’ (susceptible to race 1, without any beneficial alleles for FLS resistance) were used to investigate the expression patterns of the selected potential candidates by quantitative RT-PCR (qRT-PCR) analysis in order to ensure that the candidate gene transcription in response to FLS is directly related to the GWAS results. The leaves of each soybean accession from the FLS inoculation and the control group were collected at 0, 2, 4, 8, 12, 24, 36, 48, 60, and 72 h. The qRT−PCR assay was conducted on ABI 7500 Fast instrument by using SuperReal PreMix Plus (SYBR Green) Kit (TIANGEN, FP205). The GmActin4 (GenBank accession no. AF049106) was used as the internal standard control of soybean. The sequences of the primer pairs used for amplifying the candidate genes were listed in Supplementary Table 2.

Sun M., Na C., Jing Y., Cui Z., Li N., Zhan Y., Teng W., Li Y., Li W., Zhao X, & Han Y. (2022). Genome-Wide Association Analysis and Gene Mining of Resistance to China Race 1 of Frogeye Leaf Spot in Soybean. Frontiers in Plant Science, 13, 867713.

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Quantitative RT-PCR Protocol for Gene Expression

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Total RNA was isolated using the RNAsimple Total RNA Kit (TIANGEN) according to the manufacturer’s instructions. Reverse transcription was performed using FastQuant RT Kit (TIANGEN) according to the manufacturer’s protocol. For duplex RT-PCR, 0.5 μL cDNA from each sample was amplified with the GPRC5A primers (6 (link)) plus β-actin competition primers from Ambion in high-fidelity PCR master mix (Roche Applied Science) according to the manufacturer’s instructions. For specific quantitative real-time PCR, experiments were performed using SuperReal PreMix Plus SYBR Green Kit (TIANGEN) according to the manufacturer’s protocol. All primers used for quantitative real-time PCR are listed in Supplemental Table 1.

Song H., Ye X., Liao Y., Zhang S., Xu D., Zhong S., Jing B., Wang T., Sun B., Xu J., Guo W., Li K., Hu M., Kuang Y., Ling J., Zhang T., Wu Y., Du J., Yao F., Chin Y.E., Wang Q., Zhou B.P, & Deng J. (2023). NF-κB represses retinoic acid receptor–mediated GPRC5A transactivation in lung epithelial cells to promote neoplasia. JCI Insight, 8(1), e153976.

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Quantitative Gene Expression Analysis in M. purpureus

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Standard DNA manipulation techniques were performed as described by Green and Sambrook (2012) . Total RNA from M. purpureus was prepared using the RNAprep Pure Plant Kit (TIANGEN, Beijing, China) following the manufacturer’s instructions. RNA was subjected to reverse transcription to generate cDNA using Quantscript RT Kit (TIANGEN, Beijing, China) following the manufacturer’s protocol. Quantitative Real-Time PCR (q RT-PCR) was performed using the SuperReal PreMix Plus (SYBR Green) Kit (TIANGEN, Beijing, China) in CFX96 Touch Real-Time PCR System (Bio-Rad, Hercules, CA, United States) with the following cycling conditions: 95°C for 2 min, followed by 40 cycles of 94°C for 20 s, 63°C for 45 s, and 60°C for 5 min. The GAPDH gene was used for transcript normalization. All reactions were performed in triplicate. Data were analyzed using the 2^–ΔΔCt method corrected for primer efficiencies using the untreated group mean as the reference condition (Schmittgen and Livak, 2008 (link)). Primers used for gene transcription level assay by q RT-PCR were listed in Supplementary Table 1.

Yin S., Zhu Y., Zhang B., Huang B, & Jia R. (2022). Diverse Effects of Amino Acids on Monascus Pigments Biosynthesis in Monascus purpureus. Frontiers in Microbiology, 13, 951266.

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