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Taqman double dye probes

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Taqman double dye probes are a type of DNA detection probe used in real-time PCR (Polymerase Chain Reaction) assays. These probes are labeled with a fluorescent reporter dye and a quencher dye, which allows for the detection and quantification of target DNA sequences during the amplification process. The core function of Taqman double dye probes is to provide a sensitive and specific method for the detection and quantification of target DNA in real-time PCR applications.

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Lab products found in correlation

Precision nanoscript2 reverse transcription kit, by Primerdesign (3 mentions) Mirneasy mini kit, by Qiagen (2 mentions) Nanoscript2 kit, by Primerdesign (2 mentions) Purelink rna micro scale kit, by Thermo Fisher Scientific (2 mentions) Abi 7500 fast, by Primerdesign (2 mentions) Cfx connect real time system, by Bio-Rad (1 mentions) Universal cdna synthesis kit 2, by Qiagen (1 mentions) Exilent sybr green master mix, by Qiagen (1 mentions) Mircury lna universal rt microrna pcr primers, by Qiagen (1 mentions)

5 protocols using taqman double dye probes

1

Validating Transcriptome Data by qPCR

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Qualitative polymerase chain reaction (qPCR) was performed to confirm the reliability of RNA-seq and microarray data. cDNA was synthesized using the Precision nanoScript2 Reverse Transcription kit (PrimerDesign, Southampton, United Kingdom) for mRNAs and the Universal cDNA Synthesis kit II (Exiqon, Wobrun, Massachusetts, USA) for miRNAs. The expression of selected genes was measured using Taqman double dye probes and PrecisionPLUS MasterMix with ROX (PrimerDesign, Southampton, United Kingdom). The miRNAs qPCR reactions were performed using commercial miRCURY LNA™ Universal RT microRNA PCR primers and the ExiLENT SYBR® Green master mix (Exiqon, Wobrun, Massachusetts, USA). All experiments were performed in a light cycler CFX Connect Real-Time System (BIO-RAD, Hertfordshire, UK). The following genes–KCNA4, ACTN2, KCNK3, NANOG, TNNT2, KCNQ5, KCNJ5 and KCNB1, and miRNAs—miR-143-5p, miR-192-5p, miR-187-3p, miR-208b-3p, miR-338-5p, miR-335-5p, miR-432-5p, miR-490-5p, miR-499a-5p, miR-503-5p and miR-92b-3p, were investigated. YWHAZ and RPL13A were used as references to normalize the results, while miRNA-16-5p and miR-103a-3p were used to normalize the miRNAs results. Relative expression and FC values were calculated using the 2-ΔΔCt method.
Larupa Santos J., Rodríguez I., S. Olesen M., Hjorth Bentzen B, & Schmitt N. (2020). Investigating gene-microRNA networks in atrial fibrillation patients with mitral valve regurgitation. PLoS ONE, 15(5), e0232719.
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2

Cardiac tissue gene expression analysis in AT pigs

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Cardiac tissue from each of the four heart chambers was obtained from long-term AT pigs (n=6) or healthy controls (HC) (n=6). After premedication with zoletil pig mixture (250 mg dry tiletamin+zolazepam, 6.5 ml xylazine 20 mg/ml, 1.25 ketamine 100 mg/ml, 2.5 ml butorphanol 10 mg/ml, and 2 ml methadone 10 mg/ml) 0.1 ml/ kg given IM, the pigs were euthanized by intravenous injection of pentobarbital 200 mg/ml, and the hearts were excised and placed in ice-cold cardioplegic solution (NaCl 110.0 mmol/L, KCl 16.0 mmol/L, MgCl2 16.0 mmol/L, CaCl2 1.2 mmol/L, NaHCO3 10.0 mmol/L). Total RNA from tissue samples was extracted according to manufacturer’s instruction (miRNeasy Mini Kit, Qiagen, CA, USA). RNA was reverse transcribed to cDNA using the nanoscript2 kit (Primerdesign, UK). Expression profiling of the SK channels (KCNN1, KCNN2 and KCNN3) was performed using quantitative real-time PCR (qPCR) with Taqman double dye probes (Primerdesign, UK). For further details, please see supplemental material.
Diness J.G., Skibsbye L., Simó-Vicens R., Santos J.L., Lundegaard P., Citerni C., Sauter D.R., Bomholtz S.H., Svendsen J.H., Olesen S.P., Sørensen U.S., Jespersen T., Grunnet M, & Bentzen B.H. (2017). Termination of Vernakalant-Resistant Atrial Fibrillation by Inhibition of Small-Conductance Ca2+-Activated K+ Channels in Pigs. Circulation. Arrhythmia and Electrophysiology, 10(10), e005125.
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3

Cardiac SK Channel Expression Profiling

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Cardiac tissue from each of the 4 heart chambers was obtained from long-term AT pigs (n=6) or healthy controls (HC; n=6). After pre-medication with zoletil pig mixture (250 mg dry tiletamin+zolazepam, 6.5 mL xylazine 20 mg/mL, 1.25 ketamine 100 mg/mL, 2.5 mL butorphanol 10 mg/mL, and 2 mL methadone 10 mg/mL) 0.1 mL/ kg given intramuscularly, the pigs were euthanized by intravenous injection of pentobarbital 200 mg/mL, and the hearts were excised and placed in ice-cold cardioplegic solution (NaCl 110.0, KCl 16.0, MgCl2 16.0, CaCl2 1.2, and NaHCO3 10.0 mmol/L). Total RNA from tissue samples was extracted according to manufacturer’s instruction (miRNeasy Mini Kit, Qiagen, CA). RNA was reverse transcribed to cDNA using the nanoscript2 kit (Primerdesign, United Kingdom). Expression profiling of the SK channels (KCNN1, KCNN2, and KCNN3) was performed using quantitative real-time quantitative polymerase chain reaction with Taqman double dye probes (Primerdesign, United Kingdom). For further details, please see Data Supplement.
Diness J.G., Skibsbye L., Simó-Vicens R., Santos J.L., Lundegaard P., Citerni C., Sauter D.R., Bomholtz S.H., Svendsen J.H., Olesen S.P., Sørensen U.S., Jespersen T., Grunnet M, & Bentzen B.H. (2017). Termination of Vernakalant-Resistant Atrial Fibrillation by Inhibition of Small-Conductance Ca2+-Activated K+ Channels in Pigs. Circulation. Arrhythmia and Electrophysiology, 10(10), e005125.
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4

qPCR Analysis of Gene Expression

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Prior to cell lysis cells were washed with PBS three to four times. RNA was extracted using the PureLink RNA Micro Scale Kit (Invitrogen) that isolates large RNAs. Following isolation RNA integrity was checked on 1 % native agarose gels and confirmed to be intact, while RNA purity was confirmed via Nanodrop 2000 to be high. Reverse transcription was performed on 100 ng of total RNA using oligodT primers part of the Precision nanoScript2 Reverse Transcription Kit (Primerdesign) following the manufacturer’s instructions. Following formation of cDNA qPCR was run on the ABI 7500 fast machine using double-dye Taqman probes (Primerdesign) and the Taqman Fast Universal PCR Master Mix (2×), no AmpErase UNG (Applied Biosystems) according to the manufacturer’s instructions. Changes in mRNA expression were calculated using the 2—ΔΔCT method, while beta actin (ACTB) served as the internal normalization gene. The amplification efficiency of the double-dye Taqman assays was further confirmed to be ≈100%, which justified the use of the 2—ΔΔCT method.
Pop M.A, & Almquist B.D. (2021). Controlled Delivery of MicroRNAs into Primary Cells Using Nanostraw Technology. Advanced nanobiomed research, 1(6), 2000061.
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5

qPCR Analysis of Gene Expression

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Prior to cell lysis cells were washed with PBS three to four times. RNA was extracted using the PureLink RNA Micro Scale Kit (Invitrogen) that isolates large RNAs. Following isolation RNA integrity was checked on 1 % native agarose gels and confirmed to be intact, while RNA purity was confirmed via Nanodrop 2000 to be high. Reverse transcription was performed on 100 ng of total RNA using oligodT primers part of the Precision nanoScript2 Reverse Transcription Kit (Primerdesign) following the manufacturer’s instructions. Following formation of cDNA qPCR was run on the ABI 7500 fast machine using double-dye Taqman probes (Primerdesign) and the Taqman Fast Universal PCR Master Mix (2×), no AmpErase UNG (Applied Biosystems) according to the manufacturer’s instructions. Changes in mRNA expression were calculated using the 2—ΔΔCT method, while beta actin (ACTB) served as the internal normalization gene. The amplification efficiency of the double-dye Taqman assays was further confirmed to be ≈100%, which justified the use of the 2—ΔΔCT method.
Pop M.A, & Almquist B.D. (2021). Controlled Delivery of MicroRNAs into Primary Cells Using Nanostraw Technology. Advanced nanobiomed research, 1(6), 2000061.
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