Procedure was performed as previously described [21 (link)]. Briefly, 293T cells were seeded in five 10cm culture dishes and transfected with ADAR1 or Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) constructs together with miR-222 precursor construct. After 48 h, RNA was extracted from one culture dish using Tri Reagent (Sigma-Aldrich) in order to assess ADAR1, CEACAM1 and pri-miR-222 expression by qRT-PCR as described above. The remaining cells were immunoprecipitated using Dynabeads Protein G (Invitrogen, cat#Dy_10003D) and anti-ADAR (Sigma-Aldrich) or anti-CEACAM1 (MRG1 [57 (link)]) antibodies. At the end of the precipitation procedure, RNA was extracted using miRNeasy Kit (Qiagen, cat#217004). Reverse transcription was obtained using Universal Transcriptor cDNA master (Roche). Successful transfection and immunoprecipitation were confirmed by WB. Pri-miR-222 expression following immunoprecipitation was assessed by qRT-PCR.
Transcriptor universal cdna master
Manufactured by Roche
Sourced in United States, Germany, Switzerland, Poland
Transcriptor Universal cDNA Master is a reverse transcription reagent that enables the conversion of RNA into complementary DNA (cDNA). It provides a convenient and efficient way to generate cDNA from various RNA sources, including total RNA, poly(A)+ RNA, and in vitro transcribed RNA.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (10 mentions)
Lightcycler 480 sybr green 1 master, by Roche (8 mentions)
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
High pure rna isolation kit, by Roche (7 mentions)
Faststart universal sybr green master, by Roche (7 mentions)
Tri reagent, by Molecular Research Center (7 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (7 mentions)
Tri reagent, by Merck Group (5 mentions)
Rneasy plus mini kit, by Qiagen (5 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (5 mentions)
Lightcycler 480 instrument 2, by Roche (5 mentions)
Lightcycler 96, by Roche (5 mentions)
Trizol regent, by Thermo Fisher Scientific (4 mentions)
Lightcycler 96 system, by Roche (4 mentions)
Lightcycler 480, by Roche (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (4 mentions)
Universal probe library assay design center, by Roche (3 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
88 protocols using transcriptor universal cdna master
1
Assessing miR-222 Regulation by ADAR1 and CEACAM1
2
Total RNA Isolation and cDNA Synthesis
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Total RNA was isolated from melanoma lines, melanocytes and primary cultures using Tri Reagent (Sigma-Aldrich, Rehovot, Israel), and cDNA was generated by Universal Transcriptor cDNA master (Roche Diagnostics, Basel, Switzerland), according to the manufacturer’s instructions.
3
Comprehensive RNA Extraction and Reverse Transcription
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Total RNA was isolated using Tri Reagent (Sigma-Aldrich, cat#T9424), and cDNA was generated by High-capacity reverse transcriptase kit (Applied Biosystems, cat#4374966) using random hexamer primers or Universal Transcriptor cDNA master (Roche Diagnostics, cat#05893151001), according to manufacturer's instructions. cDNA for miRNAs was generated using TaqMan microRNA custom primers (Applied Biosystems) or Universal cDNA synthesis kit (Exiqon, cat#203301).
4
Total RNA Extraction and cDNA Synthesis
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Total RNA was isolated using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer's instructions. cDNA was generated by Transcriptor Universal cDNA Master (Roche, Penzberg, Germany).
5
RNA Extraction and Real-Time PCR
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Total RNA was extracted by using TRIzol Regent (Invitrogen). The RNA was reverse transcribed using a Transcriptor Universal cDNA Master (Roche) according to the manufacturer's instructions. Real-time PCR was performed as described previously (Cong et al., 2016 ). See Supplemental Information for primer sequences.
6
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted using RNeasy Plus Mini Kit (Qiagen) in accordance with the manufacturer’s protocol. 500 ng of total RNA was subjected to reverse transcription using Transcriptor Universal cDNA Master (Roche). RT–qPCR reactions were set up in duplicates using PowerUp SYBR Green Matermix (Thermo Fisher) and run on a QuantStudio 6 Flex instrument (Thermo Fisher). Relative quantitation was performed by a delta Ct method with normalization to housekeeping gene RPLP0. All primer sequences are listed in Table S3 .
7
Total RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted with ISOGEN (Nippon Gene, Tokyo, Japan) and cDNA was generated using transcriptor universal cDNA master (Roche Diagnostics GmbH, Mannheim, Germany). The expression levels of mRNA were quantified by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) using commercial primer-probe pairs (Applied Biosystems, Foster City, CA).
8
Plasmid Generation and Characterization
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Plasmids used in this study are listed in Supplementary Table 3 . cDNAs were made using Transcriptor Universal cDNA Master (Roche Applied Science, 05 893 151 001). PCR products were amplified from cDNA using Phusion high fidelity DNA polymerase with primers containing the Gateway recombination site or restriction enzyme sites for Gateway entry vector and cloned into pDONR221 or pENTR using Gateway recombination cloning. Plasmids were made by conventional restriction enzyme-based cloning and/or by use of the Gateway recombination system (Invitrogen). Point-mutants were generated using the QuikChange site-directed mutagenesis (Stratagene, 200523). Oligonucleotides for mutagenesis, PCR and DNA sequencing were from Invitrogen or Sigma. Plasmid constructs were verified by conventional restriction enzyme digestion and/or by DNA sequencing with BigDye (Applied Biosystems, 4337455).
9
DDR1 Inhibition Modulates Collagen-Induced PEC
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Mouse primary PEC were plated and rested overnight. Cells were incubated in RPMI-1640 containing 1% serum and supplemented with different concentrations of DDR1 inhibitor (0.01, 0.1 and 1 μM) for 1 h followed by additional treatment with type I collagen 100 μg/mL (Nitta Gelatin, Japan). After 6 or 24 h of type I collagen stimulation, cells were lysed for phospho DDR1 ELISA (Cell Signaling Technology) and total RNA isolation. Total RNA was isolated and amplified using an RNeasy Mini kit (Qiagen) and Transcriptor Universal cDNA Master (Roche) according to manufacturer’s instructions. Quantitative RT-PCR was performed on a LightCycler LC480 (Roche) for C3, Mmp2, Mmp14 and Vcam1 using the following primers: C3 (Mm01232779_m1, TaqMan® Gene Expression Assays, Applied Biosystems), Mmp2 (Mm00439498_m1), Mmp14 (Mm00485054_m1), and Vcam1 (Mm01320970_m1). Relative gene expression was calculated with the 2-∆Ct method using GAPDH as an endogenous control.
10
Cisplatin-induced gene expression
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A2780-EV and A2780-COMMD1 cells were grown to 70% confluency and were left untreated or were treated with 2mM cisplatin for 24hrs. Cells were harvested in QIAzol Lysis Reagent (Qiagen), and total RNA was isolated by chloroform extraction. Isopropanol-precipitated and ethanol-washed RNA pellets were dissolved in RNase/DNase free water. One microgram of RNA was used to prepare cDNA with the Transcriptor Universal cDNA Master (Roche), according to the protocol provided by the manufacturer. 20 ng cDNA was used for subsequent quantitative real-time PCR (qRT-PCR) analysis using FastStart SYBR Green Master (Roche) and 7900HT Fast Real-Time PCR System (Applied Biosystems). The following PCR program was used: 50°C/2 minutes, 95°C/10 minutes, 40 cycles of 95°C/15 seconds and 60°C/1 minute. Expression data were analyzed using SDS 2.3 software (Applied Biosystems), using the ‘standard curve’ method of calculation. GAPDH expression was used as an internal control. Primer sequences are listed in S1 Table .
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