Total RNA was isolated from RD cells at 3, 24, or 96 h after radiation exposure using TRIsureTM (BIOLINE, London, UK) according to the manufacturer’s instructions. cDNA was obtained and analysed as previously described [15 (link)]. Transcript levels of DNMT3A, DNMT3B, and MYHC genes were quantified using specific TaqMan Real-Time Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). The mRNA expression levels of CDKN1A, MYOD and MYOGENIN were analysed with the SensiFAST SYBR Hi-ROX Kit (BIOLINE). All samples were normalised according to GAPDH transcript levels. Expression levels of human miR-106b, miR-17-5p, miR-133a, and miR-206 were quantified using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) and miRNA-specific TaqMan MGB primers/probe (Life Technologies, Carlsbad, CA, USA). U6 small nuclear RNA levels were used as an internal control. The relative amount of each mRNA or miRNA was calculated by the comparative Ct method [27 (link)] and expressed as fold change in comparison to negative control cells. Each sample was run in triplicate, in at least three independent experiments.
Taqman real time gene expression assays
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan Real-Time Gene Expression Assays are a line of pre-designed and pre-optimized gene expression assays developed by Thermo Fisher Scientific. These assays utilize the TaqMan probe-based detection technology for quantitative real-time PCR analysis of gene expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Stepone v2, by Thermo Fisher Scientific (3 mentions)
Sensifast sybr hi rox kit, by Meridian Bioscience (2 mentions)
Stepone real time system, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Trisure, by Meridian Bioscience (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Taqman mgb primers probe, by Thermo Fisher Scientific (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using taqman real time gene expression assays
1
Radiation-Induced Gene and miRNA Expression
2
Quantitative Analysis of EPHA2 and EFNA1 mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated by tumour tissues ground under liquid nitrogen using 1 ml of TRIzol LS reagent (Invitrogen, Carlsbad, CA) per 50–100 mg of sample according to the manufacturer’s protocol. RNA concentration and purity were measured by NanoDrop 2000 (Thermo Fisher Scientific, Inc., Waltham, MA). Total RNA (2 μg) was subjected to reverse transcription with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Quantitative real-time PCR (Q-PCR) for human EPH-A2 (Hs00171656) and Ephrin-A1 (Hs00358886) mRNAs was performed using the specific TaqMan Real Time Gene Expression Assays (Applied Biosystems). Each sample was run in triplicate, in at least two independent experiments, on a StepOne Real Time System (Applied Biosystems) machine. Transcript levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, used as endogenous control. Relative quantification (RQ) of each mRNA in tumour samples in comparison to NSM was calculated by the comparative Ct method (2-ΔΔCt), using the StepOne v2.3 software (Applied Biosystems). RQmax and RQmin, which are the maximum and minimum limits of the RQ values based on the standard error of the mean ΔCt values at 95% confidence interval, were used to plot error bars.
3
Quantitative mRNA and miRNA Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA, isolated from RMS tumour biopsies and cell lines, was reverse transcribed and analysed by using quantitative Real Time PCR (q-PCR) with specific TaqMan Real-Time Gene Expression Assays (Applied Biosystems), as previously described (Megiorni et al. 2016 (link)). Human PARP1 (Hs00242302_m1), PARP2 (Hs00193931_m1) and PARP3 (Hs00193946_m1) mRNA assays were used. Samples were normalized according to GAPDH transcript levels. Expression of miR-124-3p was analysed as previously described (Megiorni et al. 2014 (link)), by using sequence-specific TaqMan MicroRNA Assays (Applied Biosystems). U6 small nuclear RNA levels were used as internal control. The amount of each mRNA or miRNA was calculated by the comparative Ct method (Livak and Schmittgen 2001 (link)) and expressed as fold change using the StepOne v2.3 software (Applied Biosystems). Each sample was run in triplicate, in at least two independent experiments.
4
Quantitative Analysis of Myogenic Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA (2 μg) was subjected to reverse transcription (RT) with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Quantitative Real Time PCR (Q-PCR) for human DNMT1 (Hs00154749_m1), DNMT3A (Hs01027166_m1), DNMT3B (Hs00171876_m1) and MyHC (Hs00428600_m1) mRNAs was performed using specific TaqMan Real-Time Gene Expression Assays (Applied Biosystems). Q-PCRs for human MYOD and Myogenin transcripts was carried out with the SensiFAST SYBR Hi-ROX Kit (Bioline, London, UK). Primer sequences are available under request. All Q-PCR assays were performed on a StepOne Real Time System (Applied Biosystems) machine. Samples were normalized according to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels. RT for human miR-133a (000458) and miR-206 (000510) was carried out with TaqMan MicroRNA Assay kit (Applied Biosystems) using 20 ng of total RNA sample and the specific stem-loop primer according to manufacturer's protocols. Data were normalized to U6 small nuclear RNA (RNU6, 001093) levels. The amount of each mRNA/miRNA was calculated by the comparative Ct method and expressed as fold change using the StepOne v2.3 software (Applied Biosystems). Each sample was run in triplicate in at least three independent experiments, unless specified differently in the figures.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!