Taqman kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The TaqMan kit is a Real-Time PCR (Reverse Transcription-Polymerase Chain Reaction) assay used for the detection and quantification of target DNA or RNA sequences. The kit includes all the necessary components, including probes and primers, for performing the PCR reaction. The TaqMan technology utilizes fluorescent reporter dyes to monitor the amplification of the target sequence in real-time.

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Lab products found in correlation

63 protocols using taqman kit

Quantification of lncRNA and miRNA

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Quantification of LncRNA (TUG-1) was performed using commercially available Taqman kit supplied from Applied Biosystems according to manufacturer’s instructions (Part Number 4375575)^{19 (link)–21 (link)}. The process of qt-PCR was started and results were displayed from the cDNA for 2 different gene targets; TUG-1 and its housekeeping control Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The threshold cycle^{22 (link)} values were calculated for both target genes samples (TUG-1 and GAPDH). Delta CT values, Delta (delta CT) values and induction percentages values were calculated.
Quantification of miR-186 was performed using commercially available Taqman kit supplied from Applied Biosystems according to manufacturer’s instructions (Part Number 4364031)^{23 (link)–26 (link)}. The process of qt-PCR was started and results were displayed from the cDNA for 2 different gene targets; miR-186 and its housekeeping control U6. The CT values were calculated for both target genes samples (miR-186 and U6). Finally, Delta CT values, Delta (delta CT) values and induction percentages values were calculated.

Azzam H.N., El-Derany M.O., Wahdan S.A., Faheim R.M., Helal G.K, & El-Demerdash E. (2022). Metabolic/hypoxial axis predicts tamoxifen resistance in breast cancer. Scientific Reports, 12, 16118.

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Quantifying Gene Expression in Drosophila

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Total RNA was prepared from 20 wing imaginal discs from larvae of the following genotypes – ApGal4/+ (control), ApGal4/P[hNF2^Iso1]; P[hNF2^Iso1]/+, ApGal4/+; (2X)P[hNF2^Iso2]/+ - using Trizol (Invitrogen). cDNA was produced using the Taqman kit (ABI) and quantitative PCR performed using the LightCycler FastStart SYBR green system (Roche).

Gavilan H.S., Kulikauskas R.M., Gutmann D.H, & Fehon R.G. (2014). In Vivo Functional Analysis of the Human NF2 Tumor Suppressor Gene in Drosophila. PLoS ONE, 9(3), e90853.

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Quantifying miRNA and mRNA Levels

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Total RNA from cells was purified using Trizol (Invitrogen, USA) and reverse transcribed using a TaqMan kit (ABI, USA). The expression of miRNAs was determined using TaqMan microRNA Assay kits (ABI, USA) with U6 as a reference. The expression levels of TGF-βRI/II and SMADs were assayed by quantitative real-time PCR (qRT-PCR) using SYBR Green PCR Master Mix (Toyobo, Japan) with β-actin as a reference. The relative miRNA and mRNA levels were calculated by the standard 2^−ΔΔCt method. The primers used are listed in Supplementary Table S1.

Fang F., Huang B., Sun S., Xiao M., Guo J., Yi X., Cai J, & Wang Z. (2018). miR-27a inhibits cervical adenocarcinoma progression by downregulating the TGF-βRI signaling pathway. Cell Death & Disease, 9(3), 395.

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Quantitative Analysis of Liver IL-10 Expression

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Tissue samples representing the whole liver were obtained 10 days after gene transfer. These samples were cut into small pieces and homogenized in buffer (Promega^®, Barcelona, Spain) with an Ultra-Turrax homogenizer (Hielscher Ultrasonics GmbH, Teltow, Germany). Further purifications with Maxwell RNA and DNA purification from tissue kits (Promega^®, Barcelona, Spain) were performed before spectrophotometric quantification. RNA retrotranscription to cDNA was carried out using 1 μg total RNA (DNA free), random hexamers and a High Capacity cDNA Archive Kit (Thermo Fisher, Madrid, Spain). For quantitative real-time qPCR, TaqMan PCR master mix (Thermo Fisher, Madrid, Spain) was employed according to the instructions of the manufacturer. The specific oligonucleotides for human IL-10 employed were a pre-mixed TaqMan kit from Thermo Fisher (cat no. Hs00961622_m). Quantitative data were calculated as the number of DNA and RNA copies on a regression curve which was plotted employing the same injected plasmid containing the hIL10 gene, prepared with a known concentration of hIL-10 plasmid and serial dilutions 1/10. Linearity of the standard curve included from 10^3 to 10^7 copies with a correlation coefficient > 0.95. Data plotting was performed using R (version 3.1.2) software.

Sendra L., Herrero M.J., Montalvá E.M., Noguera I., Orbis F., Díaz A., Fernández-Delgado R., López-Andújar R, & Aliño S.F. (2019). Efficacy of interleukin 10 gene hydrofection in pig liver vascular isolated ‘in vivo’ by surgical procedure with interest in liver transplantation. PLoS ONE, 14(11), e0224568.

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Quantitative Real-Time PCR Analysis

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An amount of 200 ng total RNA was reverse transcribed using the TaqMan kit (Thermofisher). The cDNA level was analyzed on an MxPro3005 (Agilent Technologies) using a SensiFast SYBR No-ROX Kit (Bioline, UK), 1 μl cDNA and 250 nM of each primer. The qPCR was done using the following thermal profile: 95°C for 2 min, then 40 cycles of 12 s at 95°C, 7 s at 64°C and 3 s at 72°C with additional melt point analysis. Gene expression was calculated using an amplification efficiency (E) of 1.95 and values were normalized on reference gene UBE2D2 (NM_181838) expression. The sequences of the qPCR primers and the gene identifiers used are listed in the S1 Table. The students t-test was performed for statistical analysis and differences in expression for p-values < 0.05 = * (< 0.01 = **, < 0.001 = ***, < 0.0001 = ****) were regarded as significant. Paired or unpaired t-tests were performed, depending on the experimental setup, using GraphPad Prism version 6 for Mac (GraphPad Software, La Jolla, California, USA).

Kadler S., Vural Ö., Rosowski J., Reiners-Schramm L., Lauster R, & Rosowski M. (2020). Effects of 5-aza-2´-deoxycytidine on primary human chondrocytes from osteoarthritic patients. PLoS ONE, 15(6), e0234641.

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Evaluating Anti-miR21 Efficacy in Breast Cancer

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To examine the efficiency of anti-miR21, MCF7 and MCF1/ADR (10⁵ cells/well) cells were seeded in 12-well culture plates and incubated for 24 hours. The cells were then treated with HMNs containing anti-miR21 or scramble control at a final concentration of 213.3 nM for 48 hours.
Total RNA was extracted from cells with Trizol reagent (Thermo Fisher Scientific), and reverse-transcribed to complementary DNA using a TaqMan miRNA reverse-transcription kit (Takara, Kyoto, Japan). Then, real-time polymerase chain reaction (PCR) was performed on an Applied Biosystems 7500 (Thermo Fisher Scientific) using the TaqMan kit according to the standardized protocol. Both reverse transcription and PCR primers were purchased from GenePharma. miR21 expression was normalized using the

2^{- Δ Δ C_{T}}

method relative to human U6 small nuclear RNA. All reactions were performed in triplicate. The change in miR21 expression was calculated as the fold variation relative to the untreated control.

Rui M., Qu Y., Gao T., Ge Y., Feng C, & Xu X. (2016). Simultaneous delivery of anti-miR21 with doxorubicin prodrug by mimetic lipoprotein nanoparticles for synergistic effect against drug resistance in cancer cells. International Journal of Nanomedicine, 12, 217-237.

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Validating Microarray Data via RT-qPCR

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RT-qPCR assays were performed to validate the microarray data. Total RNA was extracted from A549 cells using TRIzol reagent. Single-strand cDNA was synthesized from 1 mg total RNA using the Prime-Script™ Reagent kit with gDNA Eraser (TransGen Biotech Co., Ltd.). The steps were 70°C for 5 min, 37°C for 5 min, 42°C for 60 min and 70°C for 10 min to end the reaction. To detect the mRNA expression levels of the DEGs, qPCR (TaqMan kit; Thermo Fisher Scientific, Inc.) was conducted on a Q1 PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primer sequences are listed in Table I. The following thermocycling conditions were applied: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, 60°C for 15 sec and 72°C for 10 sec and 72°C for 7 min for final extension. Data were presented as a relative average value ± SEM after normalization with the average value of the housekeeping gene GAPDH. Direct comparison was performed between the control and H₂O₂-treated groups for the same gene and 2^−ΔΔCq method was used for the relative quantification of gene expression (16 (link)).

Li Y., Wei Z., Huang S, & Yang B. (2020). mRNA expression and DNA methylation analysis of the inhibitory mechanism of H2O2 on the proliferation of A549 cells. Oncology Letters, 20(6), 288.

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Quantifying Mutant Rps2 mRNA Levels

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The relative ratios of mouse wild-type Rps2 and Rps2-A226Y mRNA were measured using Taqman RT-PCR, with primers flanking the site of mutation (forward 5′-GGT GAC AGG CCG CTG TGG CTC TGT GCT GGT-3′, reverse 5′-AAG TTG CCC AGG GTG GCA GTG CAG-3′) and TaqMan probes specific for wild-type Rps2 (5′-TGC TAC ACT TCA GCC-3′, NED) or Rps2-A226Y (5′-CTA CAC TTC ATA CAG AG-3′, FAM). Experiments were conducted using a TaqMan™ kit (Thermo Scientific, Waltham, MA, USA, 4352405) and the 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA); amplification of 40 cycles (95 °C for 20 s and 60 °C for 45 s). The 2^−ΔΔCT method was used to calculate the ratio between wild-type and mutant mRNA.

Moore J., Osinnii I., Grimm A., Oettinghaus B., Eckert A., Frank S, & Böttger E.C. (2021). Silencing of the ER and Integrative Stress Responses in the Liver of Mice with Error-Prone Translation. Cells, 10(11), 2856.

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Quantitative Methylation-Specific PCR for DNA Methylation Analysis

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DNA methylation levels in tissues and cultured cells were performed according to a method known as quantitative methylation-specific PCR, which is designed to amplify bisulfite-modified methylated DNA sequences. 27 In brief, genomic DNA from clinical tissues and cultured cells was extracted using a kit (Sigma-Aldrich; catalog number 11814770001) following the guidelines of the manufacturer. For each sample, 1 mg of purified DNA was treated with the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA; catalog number D5007) to convert cytosine to uracil. DNA was then applied to quantitative methylation-specific PCR assays using a TaqMan kit (Thermo Fisher Scientific; catalog number 4440038) with the following primers: forward, 5 0 -GAATTTGGGTTTTTATTTTTTAGG-3 0 ; and reverse, 5 0 -CCAAACCCTAAAACTAACTCTCTC-3 0 .

Yang C., Lu W., He H, & Liu H. (2020). Inflammation and DNA Methylation-Dependent Down-Regulation of miR-34b-5p Mediates c-MYC Expression and CRL4^DCAF4 E3 Ligase Activity in Colitis-Associated Cancer. The American journal of pathology, 190(3).

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Quantitative Gene Expression Analysis

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Half of each sample was used for gene expression analysis, and all samples within each treatment group were pooled. The experiment was repeated in its entirety to produce a replicates. Bioreactor constructs were transferred from storage at −80°C directly into a cryomill (SPEX SamplePrep, USA) and pulverized in liquid nitrogen. RNA was isolated from tissue homogenates using an acid guanidinium thiocyanate extraction protocol in phenol-chloroform, followed by precipitation in isopropanol. The resulting pellets were resuspended and the solutions concentrated in RNeasy spin columns (Qiagen, USA), quantitated with RiboGreen RNA reagent (Life Technologies, USA) and reverse-transcribed with a high-capacity cDNA kit (Life Technologies, USA). cDNA was pre-amplified using a validated commercial TaqMan kit (Life Technologies, USA) prior to reaction in a 7500 Real-Time PCR System (Applied Biosystems, USA) using custom TaqMan probes (Life Technologies, USA) in duplicate. A list of primers, probes and abbreviations used are included in Table 1. Reactions were quantified with the 2^-ΔΔCt method using GAPDH as a reference gene and are reported by fold-change with respect FDST.

Youngstrom D.W., Rajpar I., Kaplan D.L, & Barrett J.G. (2015). A bioreactor system for in vitro tendon differentiation and tendon tissue engineering. Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 33(6), 911-918.

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