Cells were treated with BETi compounds or vehicle (DMSO) in complete medium for up to 96 hours. Following treatment, cells were harvested, and transcripts quantified via TaqMan real-time PCR as previously described [30 (link),31 (link)]. Briefly, mRNA was isolated using mRNA Catcher™ PLUS purification kits according to the manufacturer’s instructions (Thermo Fisher). Taqman PCR assays were obtained from Applied Biosystems/Life Technologies. Real-time PCR was used to determine the abundance of the transcript of interest relative to the endogenous reference gene, cyclophilin in a duplex reaction using the RNA Ultrasense One-Step qRT-PCR kit (Thermo Fisher). Data were acquired using a ViiA-7 Real-Time PCR apparatus (Applied Biosystems/Thermo Fisher). The analysis was performed as 2(CT cyclophilin−CT transcript of interest), and results were normalized to DMSO-treated samples.
Rna ultrasense one step qrt pcr kit
Manufactured by Thermo Fisher Scientific
The RNA Ultrasense One-step qRT-PCR kit is a reagent system designed for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of RNA samples. The kit enables the efficient conversion of RNA to cDNA and the subsequent real-time amplification and detection of target sequences in a single reaction.
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5 protocols using rna ultrasense one step qrt pcr kit
1
BETi Compound Treatment and Transcript Analysis
2
Quantifying Transcript Abundance in Cells
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Following treatment, cells were harvested and transcripts were quantified via TaqMan real-time PCR as previously reported [43] (link). Briefly, mRNA was isolated using mRNA Catcher™ PLUS purification kits according to the manufacturer’s instructions (ThermoFisher). Taqman PCR assays were obtained from Applied Biosystems / Life Technologies. Real-time PCR was used to determine abundance of the transcript of interest relative to the endogenous control cyclophilin in a duplex reaction using the RNA Ultrasense One-step qRT-PCR kit (ThermoFisher). Data was acquired using a ViiA-7 Real-Time PCR apparatus (Applied Biosystems). The analysis was performed as 2^ (CTcyclophilin – CTtranscript of interest) and results were normalized to DMSO treated samples.
3
BETi Compound Effects on Transcripts
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Cells were treated with BETi compounds or vehicle (DMSO) in complete medium for up to 96 hours.
Following treatment, cells were harvested, and transcripts quantified via TaqMan real-time PCR as previously described 30, (link)31 (link) . Briefly, mRNA was isolated using mRNA Catcher™ PLUS purification kits according to the manufacturer's instructions (Thermo Fisher). Taqman PCR assays were obtained from Applied Biosystems / Life Technologies. Real-time PCR was used to determine abundance of the transcript of interest relative to the endogenous reference gene, cyclophilin in a duplex reaction using the RNA Ultrasense One-step qRT-PCR kit (Thermo Fisher). Data was acquired using a ViiA-7 Real-Time PCR apparatus (Applied Biosystems). The analysis was performed as 2^ (C T cyclophilin -C T transcript of interest) and results
were normalized to DMSO treated samples.
Following treatment, cells were harvested, and transcripts quantified via TaqMan real-time PCR as previously described 30, (link)31 (link) . Briefly, mRNA was isolated using mRNA Catcher™ PLUS purification kits according to the manufacturer's instructions (Thermo Fisher). Taqman PCR assays were obtained from Applied Biosystems / Life Technologies. Real-time PCR was used to determine abundance of the transcript of interest relative to the endogenous reference gene, cyclophilin in a duplex reaction using the RNA Ultrasense One-step qRT-PCR kit (Thermo Fisher). Data was acquired using a ViiA-7 Real-Time PCR apparatus (Applied Biosystems). The analysis was performed as 2^ (C T cyclophilin -C T transcript of interest) and results
were normalized to DMSO treated samples.
4
Quantitative Real-Time PCR Assay
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Relative mRNA expression was determined by TaqMan based real-time PCR, according to established protocols [39 (link)]. Adherent cells, seeded for a minimum of 24 hours (h) were lysed in situ, and RNA was isolated using Catcher PLUS kits (Life Technologies, Waltham, MA, USA). Real-time PCR was used to determine the abundance of each transcript relative to the endogenous control cyclophilin A (gene symbol PPIA) using the RNA Ultrasense One-step qRT-PCR kit (Life Technologies). Data was acquired using a ViiA-7 Real-Time PCR apparatus (Applied Biosystems Waltham, MA, USA/Life Technologies). Analysis was performed as 2(CT cyclophilin–CT marker) and results were normalized to DMSO treated samples, as specified in figures. TaqMan assay (Life Technologies) ID numbers are listed in Table S2 . Dose–response curves were prepared using an eight-point dilution scale ranging from 0.02–50 µM for apabetalone, and from 0.001–3.0 µM for JQ1.
5
Apabetalone Modulates Gene Expression
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mRNA was isolated from THP-1 cells and HUVEC cells, pretreated with apabetalone (5 or 20 uM) or vehicle (DMSO) for 1 h before stimulation with TNFα (10 ng/ml) in the presence of apabetalone or DMSO for 1 h (Additional file 1 : Figure S1) or 4 h (all other experiments) (Catcher PLUS kits; Life Technologies). Taqman PCR assays were obtained from Applied Biosystems/Life Technologies. Real-time PCR was used to determine the abundance of the transcript relative to the endogenous control cyclophilin in the same sample using the RNA Ultrasense One-step qRT-PCR kit (Life Technologies). Data was acquired using a ViiA-7 Real-Time PCR apparatus (Applied Biosystems). Analysis was performed as 2^ (CT cyclophilin – CT marker) and results were normalized to DMSO treated samples.
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