Gene expression was measured using the TaqMan kit. Briefly, the reactions were performed in 10 μl amounts including 50 ng cDNA, 5 μl KAPA PROBE FAST qPCR Kit Master Mix ABI Prism (Kapa Biosystems) and 0.5 μl appropriate TaqMan Gene Expression Assay (20×). Specific Pre-made TaqMan assays were used in this study: adenylate cyclase activating polypeptide 1 (pituitary) receptor type I (ADCYAP1R1, Applied Biosystems code-Hs01027974_m1), vasoactive intestinal peptide receptor 2 (VIPR2, Hs00173643_m1), and beta actin (ACTB, Hs01060665_g1) as the endogenous control. TaqMan PCR assays were performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems) in FastGene Fast 96 well PCR plates (Nippon Genetics Europe GmbH). The following thermal cycling specifications were performed: 20 s at 95 °C and 40 cycles each for 3 s at 95 °C and 30 s at 60 °C. Real-time PCR data was analyzed using the 2−ΔΔCt method [22 (link)].
Kapa probe fast qpcr kit master mix abi prism
Manufactured by Roche
Sourced in Poland
The KAPA PROBE FAST qPCR Kit Master Mix ABI Prism is a pre-formulated reagent designed for quantitative polymerase chain reaction (qPCR) analysis on Applied Biosystems (ABI) real-time PCR instruments. It is optimized for rapid, sensitive, and accurate detection of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
7900ht fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Revertra ace, by Toyobo (1 mentions)
Isogen, by Nippon Gene (1 mentions)
Multi beads shocker, by Yasui Kikai (1 mentions)
Rotor gene 3000 real time pcr system, by Qiagen (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Taqman primer probe, by Thermo Fisher Scientific (1 mentions)
Fastgene fast 96 well pcr plates, by Nippon Genetics (1 mentions)
5 protocols using kapa probe fast qpcr kit master mix abi prism
1
Quantifying Gene Expression via TaqMan qPCR
2
Real-Time PCR Analysis of hiPSC-Derived Particles
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hiPSC‐derived particles were homogenized using a multibeads shocker (Yasui kikai). Total RNA was isolated using the Isogen (Nippon Gene) and RNeasy Mini Kit (Qiagen) with the RNase‐Free DNase Set (Qiagen) according to the manufacturer's instructions. A total of 500 ng RNA was used as a template for the cDNA synthesis using ReverTra Ace (TOYOBO). Real‐time PCR was performed in a Step One system (ABI) using a KAPA PROBE FAST qPCR kit Master Mix ABI prism (KAPA BIOSYSTEMS). The primers used were GAPDH: Hs03929097_g1, OCT4 (POU5F1): Hs01654807_s1, NANOG: Hs04260366_g1, LIN28A: Hs00702808_s1, SOX9: Hs01001343_g1, COL2A1: Hs00264051_m1, and ACAN: Hs00153936_m1 (ABI). The amplified products were used to derive standard curves for quantitative real‐time PCR. The mRNA expression levels were normalized to the level of the GAPDH expression.
3
Quantitative RT-PCR Analysis of TP53 and MGMT
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Total RNA (500 ng) was used for cDNA synthesis. We used a QuantiTect Reverse Transcription Kit (Qiagen) and followed the manufacturer’s protocol. The cDNA samples were kept frozen at –20°C.
mRNA expression levels were measured using standard TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA): tumor protein TP53 (TP53, Hs01034249_m1), methylguanine-DNA methyltransferase (MGMT, Hs01037698_m1), and glyceraldehydes-3-phosphate dehydrogenase (GAPDH, Hs99999905_m1) as the endogenous control. TaqMan PCR assays were performed in 10-μl reactions using 50 ng cDNA, 5 μl KAPA PROBE FAST qPCR Kit Master Mix ABI Prism (Kapa Biosystems), and 0.5 μl of the appropriate TaqMan Gene Expression Assay. All reactions were run in duplicate on a Rotor Gene 3000 Real-Time PCR System (Corbett Life Science) according to the following thermal cycling conditions: 10 min at 95°C and 40 cycles each of 10 s at 95°C and 60 s at 60°C.
TP53 and MGMT expression levels were calculated using the 2–ΔCt method. ΔCt was calculated by subtracting the Ct of the investigated gene from the Ct of the endogenous control. Samples were excluded from further analysis if the Ct difference between duplicates was greater than 1 (treated as experiment error, e.g., degraded RNA). All patients with good quality RNA available (n = 46, 3 patients were found to have degraded RNA) were included in this analysis.
mRNA expression levels were measured using standard TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA): tumor protein TP53 (TP53, Hs01034249_m1), methylguanine-DNA methyltransferase (MGMT, Hs01037698_m1), and glyceraldehydes-3-phosphate dehydrogenase (GAPDH, Hs99999905_m1) as the endogenous control. TaqMan PCR assays were performed in 10-μl reactions using 50 ng cDNA, 5 μl KAPA PROBE FAST qPCR Kit Master Mix ABI Prism (Kapa Biosystems), and 0.5 μl of the appropriate TaqMan Gene Expression Assay. All reactions were run in duplicate on a Rotor Gene 3000 Real-Time PCR System (Corbett Life Science) according to the following thermal cycling conditions: 10 min at 95°C and 40 cycles each of 10 s at 95°C and 60 s at 60°C.
TP53 and MGMT expression levels were calculated using the 2–ΔCt method. ΔCt was calculated by subtracting the Ct of the investigated gene from the Ct of the endogenous control. Samples were excluded from further analysis if the Ct difference between duplicates was greater than 1 (treated as experiment error, e.g., degraded RNA). All patients with good quality RNA available (n = 46, 3 patients were found to have degraded RNA) were included in this analysis.
4
Quantitative Gene Expression Analysis
5
Quantitative Gene Expression Analysis
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TaqMan gene expression experiments were performed in 10 µL reactions including 20 ng cDNA, 5 µL KAPA PROBE FAST qPCR Kit Master Mix ABI Prism (Kapa Biosystems; Sigma-Aldrich, Poznan, Poland), and 0.5 µL appropriate TaqMan Gene Expression Assay (20×). Specific Pre-made TaqMan assays (Thermo Scientific™) B-cell CLL/lymphoma 2 (BCL2, Hs00608023_m1), BCL2-associated X protein (BAX, Hs00180269_m1), and β-actin (ACTB, Hs01060665_g1) as the endogenous control were used in this study. TaqMan PCR assays were performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Warszawa, Poland) in FastGene Fast 96-well PCR plates (Nippon Genetics Europe GmbH, Dueren, Germany). The following thermal cycling specifications were performed: 20 s at 95°C and 40 cycles each for 3 s at 95°C and 30 s at 60°C. Expression values were calculated using Sequence Detection System 2.3 Software. Fold induction values (RQ) were calculated according to the equation 2 -ΔΔCt , where ΔCt represents the differences in cycle threshold numbers between the target gene and β-actin, and ΔΔCt represents the relative change in these differences between examined and control cells (calibrator).
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