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Quantitect probe one step rt pcr kit

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The QuantiTect Probe One-Step RT-PCR Kit is a reagent kit designed for real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. It provides the necessary components for the detection and quantification of RNA targets.

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Lab products found in correlation

Taqman human gapdh control reagents, by Thermo Fisher Scientific (4 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions) Rneasy mini kit, by Qiagen (3 mentions) Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions) Taqman probe, by Thermo Fisher Scientific (2 mentions) 7300 real time pcr system, by Thermo Fisher Scientific (1 mentions) Taqman ribosomal rna control reagent, by Thermo Fisher Scientific (1 mentions) Eukaryotic 18s rrna endogenous control, by Thermo Fisher Scientific (1 mentions) Abi 7000 sequence detection system, by Thermo Fisher Scientific (1 mentions) Primescript rt master mix, by Takara Bio (1 mentions) Primestar max dna polymerase, by Takara Bio (1 mentions) Rneasy plus mini kit, by Qiagen (1 mentions) Systat 13, by Grafiti LLC (1 mentions) Lightcycler480 2 sequence detection system, by Roche (1 mentions) Brca2, by Thermo Fisher Scientific (1 mentions) Magattract virus mini kit, by Qiagen (1 mentions) 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions) Bioanalyzer 2100 nano chip, by Agilent Technologies (1 mentions) Aurum total rna fatty and fibrous tissue kit, by Bio-Rad (1 mentions) Rotor gene 6000 instrument, by Qiagen (1 mentions)

Spelling variants of this product

QuantiTect (227 citations) QuantiTect RT kit (187 citations) QuantiTect kit (107 citations) RT-PCR kit (45 citations) QuantiTect Probe RT-PCR (16 citations) QuantiTect Probe kit (4 citations)

11 protocols using quantitect probe one step rt pcr kit

1

Quantitative RNA Expression Analysis

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Total RNA was isolated from cells using an RNeasy Mini Kit with DNase I treatment (Qiagen), according to the manufacturer's instructions. RNA concentration was determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). In the spike experiments, 253G1 cells and primary human cardiomyocytes (PromoCell) were mixed at a defined cell number before RNA isolation. qRT-PCR was performed with the QuantiTect Probe one-step RT-PCR Kit (Qiagen) on a 7300 Real-Time PCR System (Applied Biosystems). Total RNA (50 ng per sample) was used for the analysis. The expression levels of target genes were normalized to those of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) transcript or ribosomal RNA, which were quantified using TaqMan human GAPDH control reagents (Life Technologies) or TaqMan ribosomal RNA control reagents (Life Technologies). Probes labeled with 5′-FAM/3′-TAMRA and primers were obtained from Sigma–Aldrich. The sequences of primers and probes used in the present study are listed in Table S1. All qRT-PCR reactions were run at 45 cycles.
Kuroda T., Yasuda S., Matsuyama S., Tano K., Kusakawa S., Sawa Y., Kawamata S, & Sato Y. (2015). Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells. Regenerative Therapy, 2, 17-23.
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2

qRT-PCR for Gene Expression Analysis

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Total RNA was treated with DNase I and isolated using RNeasy Mini Kit (Qiagen Hilden, Germany) according to the manufacturer's instructions. Quantitative RT-PCR was performed using the QuantiTect Probe one-step RT-PCR Kit (Qiagen) on StepOnePlus Real Time PCR system (Life Technologies). Gene expression levels were normalized to GAPDH expression levels, which were quantified using TaqMan human GAPDH control reagents (Life Technologies). Primers and probes were obtained from Sigma-Aldrich. The sequences of primers and probes are listed in Table S1.
Tano K., Yasuda S., Kuroda T., Saito H., Umezawa A, & Sato Y. (2014). A Novel In Vitro Method for Detecting Undifferentiated Human Pluripotent Stem Cells as Impurities in Cell Therapy Products Using a Highly Efficient Culture System. PLoS ONE, 9(10), e110496.
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3

Quantitative RT-PCR for Gene Expression

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Total RNA was isolated as described above. qRT-PCR was performed with the QuantiTect Probe One-Step RT-PCR Kit (Qiagen) on a StepOnePlus Real-Time PCR System (Applied Biosystems). The expression levels of target genes were normalized to those of the GAPDH transcript or 18S rRNA, which were quantified using TaqMan human GAPDH control reagents (Applied Biosystems) or eukaryotic 18S rRNA endogenous control (Applied Biosystems), respectively. Probes and primers were obtained from Sigma-Aldrich. The sequences of the primers and probes used in the present study are listed in Supplementary Table 3.
Kuroda T., Yasuda S., Tachi S., Matsuyama S., Kusakawa S., Tano K., Miura T., Matsuyama A, & Sato Y. (2019). SALL3 expression balance underlies lineage biases in human induced pluripotent stem cell differentiation. Nature Communications, 10, 2175.
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4

Quantitative RT-PCR Gene Expression Analysis

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Total RNA was extracted from cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. qRT-PCR was performed using the QuantiTect Probe One-step RT-PCR Kit (Qiagen) on the StepOnePlus Real Time PCR System (Life Technologies, Carlsbad, CA, USA). Gene expression levels were normalized to GAPDH expression levels, which were quantified using TaqMan Human GAPDH Control Reagents (Life Technologies). Primers and probes were obtained from Sigma-Aldrich. The sequences of primers and probes are listed in Table S1.
Kono K., Sawada R., Kuroda T., Yasuda S., Matsuyama S., Matsuyama A., Mizuguchi H, & Sato Y. (2019). Development of selective cytotoxic viral vectors for concentration of undifferentiated cells in cardiomyocytes derived from human induced pluripotent stem cells. Scientific Reports, 9, 3630.
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5

RNA Extraction and Gene Expression Analysis

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Total RNA was isolated with the RNeasy plus Mini Kit (Qiagen) in accordance with the manufacturer’s instructions. Contaminating genomic DNA was removed using a gDNA Eliminator spin column. cDNA was generated from 50 ng of total RNA using PrimeScript RT Master Mix (Takara Bio) and PrimeSTAR MAX DNA Polymerase (TaKaRa Bio). Real-time PCR was then performed with an ABI 7000 Sequence Detection System (Applied-Biosystems) and SYBR-green in accordance with the manufacturer’s instruction. Gene expression levels were normalized to that of GAPDH. qRT-PCR was performed using the QuantiTect Probe one-step RT-PCR Kit (Qiagen). The expression levels of target genes were normalized to those of the RNase P transcript, which were quantified using TaqMan human RNase P control reagents (Applied Biosystems). All qRT-PCR reactions were run for 45 cycles. The sequences of primers and probes used in the present study are listed in Table S1.
Kanemura H., Go M.J., Shikamura M., Nishishita N., Sakai N., Kamao H., Mandai M., Morinaga C., Takahashi M, & Kawamata S. (2014). Tumorigenicity Studies of Induced Pluripotent Stem Cell (iPSC)-Derived Retinal Pigment Epithelium (RPE) for the Treatment of Age-Related Macular Degeneration. PLoS ONE, 9(1), e85336.
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6

Gene Expression Analysis of hiPSCs

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Total RNA was isolated from hiPSCs or differentiated cells using the RNeasy Mini Kit (Qiagen) and treated with DNase I according to the manufacturer’s instructions. qRT-PCR was performed using a QuantiTect Probe One-Step RT-PCR Kit (Qiagen) on a STEPONEPLUS Real-Time PCR System (Applied Biosystems). The expression levels of target genes were normalized to those of the GAPDH transcript or 18S rRNA, which were quantified using TaqMan human Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control reagents (Applied Biosystems) or eukaryotic 18S rRNA endogenous controls (Applied Biosystems), respectively. The probes and primers were obtained from Sigma-Aldrich. The used primer and probe sequences are listed in Supplementary Table 2. PCA was performed using SYSTAT 13 software (Systat Software Inc.) after data standardization (z-scoring) for each NS/PC marker gene.
Kuroda T., Yasuda S., Matsuyama S., Miura T., Sawada R., Matsuyama A., Yamamoto Y., Morioka M.S., Kawaji H., Kasukawa T., Itoh M., Akutsu H., Kawai J, & Sato Y. (2024). ROR2 expression predicts human induced pluripotent stem cell differentiation into neural stem/progenitor cells and GABAergic neurons. Scientific Reports, 14, 690.
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7

Quantitative RT-PCR for Piscine Orthoreovirus

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The RT-qPCR was performed using the Quantitect Probe OneStep RT-PCR kit from Qiagen. For head kidney samples, the amount of total RNA was standardized to 100 ng per reaction (5 μL of 20 ng/uL). For plasma samples 5 μL total RNA was used in each reaction. Prior to one-step RT-qPCR, the template dsRNA was denatured at 95 °C for 5 min and cooled immediately on ice. The RT-qPCR targeted PRV gene segment S1 and was performed as previously described [40 (link)]. The coding regions of the revived sample from 1997 (NOR-1997) were amplified by PCR using primers shown in Table S1 and the PCR products were sequenced by Sanger sequencing (GATC Biotech AG, Konstanz, Germany).
Dhamotharan K., Tengs T., Wessel Ø., Braaen S., Nyman I.B., Hansen E.F., Christiansen D.H., Dahle M.K., Rimstad E, & Markussen T. (2019). Evolution of the Piscine orthoreovirus Genome Linked to Emergence of Heart and Skeletal Muscle Inflammation in Farmed Atlantic Salmon (Salmo salar). Viruses, 11(5), 465.
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8

Quantification of DNA Repair Gene Expression

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Total RNA was isolated from cells in 96-well plates using the Qiagen FastLane Cell Probe Kit (QIAGEN, 216413), according to the manufacturer's instructions to a final volume of 40 μL per well, at the indicated timepoint after treatment. Gene expression was evaluated by qPCR using the ONE-step QuantiTect Probe RT-PCR Kit (Qiagen, catalog no./ID: 204445). For each reaction, 2 μL of RNA were used. Real-time qPCR reactions were performed on a Roche Lightcycler 480 II Sequence Detection System.
The following Taqman probes were obtained from Thermo Fisher Scientific: BRCA2 (Hs00609073_m1), IPO8 (Hs00914057_m1), RAD51B (Hs01568768_m1), RAD54L (Hs00941668.m1), Actin (Hs01060665.g1).
For data analysis, ΔCt was calculated by subtracting average Ct of housekeeping genes from each Ct. An average ΔCt for the control group was calculated and subtracted from ΔCt to calculate negative ΔΔCt. 2−ΔΔCt was used to calculate fold change.
Jamal K., Galbiati A., Armenia J., Illuzzi G., Hall J., Bentouati S., Barrell D., Ahdesmäki M., O'Connor M.J., Leo E, & Forment J.V. (2022). Drug–gene Interaction Screens Coupled to Tumor Data Analyses Identify the Most Clinically Relevant Cancer Vulnerabilities Driving Sensitivity to PARP Inhibition. Cancer Research Communications, 2(10), 1244-1254.
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9

Quantification of Viral RNA from Plasma

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Viral RNA was extracted from 200 µl plasma using the MagAttract Virus Mini kit (Qiagen, Hilden, Germany) and the M48 robotic system (Qiagen, Hilden, Germany). For quantification, 5 µl of the eluates were reverse transcribed and amplified using the one-step QuantiTect Probe RT-PCR Kit (Qiagen) and the 7500 Real Time PCR system (Applied Biosystems) according to the manufacturer’s description. The reaction mixture contained 10 µM of each oligonucleotide: LTR forward (5′-CTGGGTGTTCTCTGGTAAG-3′), LTR- reverse (5′-CAAGACTTTATTGAGGCAAT-3′), and probe (6-carboxyfluorescein-CGAACACCCAGGCTCAAGCTGG-6-carboxytetramethyl-rhodamine)64 (link). Reverse transcription was performed for 30 min at 50 °C, followed by a denaturing step at 95 °C for 10 s. Amplification was performed for 45 cycles: 15 s at 95 °C, 45 s at 55 °C, and 34 s at 72 °C. For calculation of absolute viral RNA copy numbers, a serially diluted standard RNA was reverse transcribed and amplified in parallel. Cloning and in vitro transcription of the standard RNA was done as described67 (link).
Joas S., Parrish E.H., Gnanadurai C.W., Lump E., Stürzel C.M., Parrish N.F., Learn G.H., Sauermann U., Neumann B., Rensing K.M., Fuchs D., Billingsley J.M., Bosinger S.E., Silvestri G., Apetrei C., Huot N., Garcia-Tellez T., Müller-Trutwin M., Hotter D., Sauter D., Stahl-Hennig C., Hahn B.H, & Kirchhoff F. (2018). Species-specific host factors rather than virus-intrinsic virulence determine primate lentiviral pathogenicity. Nature Communications, 9, 1371.
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10

Quantifying P. aeruginosa in Wound Biofilms

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Scabs from wounds infected with PAO1 from two mice were surgically excised at 28 d and the duplicate specimens were processed independently. Each scab was collected and stored in 1 ml of RNALater (Qiagen, Valencia, CA) at −20°C. Two separate 72 hour colony biofilms, prepared as those used for wound inoculation, were collected in the same manner. RNA was extracted using an Aurum Total RNA Fatty and Fibrous Tissue Kit (Bio-Rad, Hercules, CA). RNA quality was verified on a Bioanalyzer 2100 Nanochip (Agilent, Santa Clara, CA). Total RNA was quantified on the Nanodrop 1000 (Nanodrop Technologies, Wilmington, DE). P. aeruginosa specific rRNA from the total RNA was analyzed by RT-qPCR of the 16S rRNA. Primer sequences and concentrations for P. aeruginosa 16S rRNA were described previously (19 (link)). RT-qPCR was performed using the one-step QuantiTect Probe RT-PCR kit (Qiagen, Valencia, CA) and the Rotorgene 6000 instrument (Corbett Research, San Francisco, CA). RNA extracted from a colony biofilm was used to construct a standard curve over five orders of magnitude. Controls lacking reverse transcriptase indicated that the RNA was free from interfering levels of DNA.
James G.A., Zhao A.G., Usui M., Underwood R.A., Nguyen H., Beyenal H., deLancey Pulcini E., Hunt A.A., Bernstein H.C., Fleckman P., Olerud J., Williamson K.S., Franklin M.J, & Stewart P.S. (2016). Microsensor and transcriptomic signatures of oxygen depletion in biofilms associated with chronic wounds. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society, 24(2), 373-383.
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