RNA was extracted using PureHelix RNA extraction solution (Nanohelix, inc., Daejeon, Korea) from kidney and lung lysate. One microgram of RNA was used for cDNA synthesis using the DiaStar RT Kit (DR23-R10k, SolGent, Inc., Daejeon, Korea). Quantitative real-time PCR (qRT–PCR) was performed using LightCycler 480 SYBR Green I Master mix and the LightCycler machine (Roche Applied Sciences, Basel, Switzerland). Murine qRT–PCR primer sequences were 5′-ACAAACGCATTGCCTGTGAGG AAG-3′ and 5′-TTTGGCTTCTGGCTTCTCCTCCTT-3′ for HDAC1; 5′-TAGGCCTCATAAAGCCACTGCTGA-3′ and 5′-ACCGGACAATCTTCTCCGACGTTA-3′ for HDAC2; 5′-TTCGAGTTCTGCTCCCGTTACACA-3′ and 5′-TAGCAGAAGCCAGAGGCCTCAAAT-3′ for HDAC3; 5′-AACCCTGAGACAAGAGTGCCAGTT-3′ and 5′-TCAGTTGCTCTCTGATGGCATGGA-3′ for HDAC6; 5′-CTCGAACTCAAAGCAGGAGAG-3′ and 5′-GTAGATGGCGAACAGG-AAGG-3′ for angiotensinogen; 5′-GAACGAATCCCGCTC-AAGA A-3′ and 5′-AGGAAGGCCTCTTTGTGAATAC-3′ for renin; 5′-GACAGGTT-CGTGGAAGAGTATG-3′ and 5′-TTGCTGCCCTCTATGGTAATG-3′ for ACE; 5′-GTAACCCGTTGAACCCCATT-3′ and 5′-CCATCCAATCGGTAGTA-GCG-3′ for 18S rRNA, sense and antisense, respectively.
Lightcycler machine
Manufactured by Roche
Sourced in Switzerland
The LightCycler is a real-time PCR (polymerase chain reaction) instrument manufactured by Roche. It is designed for the amplification and detection of nucleic acid sequences in samples. The LightCycler utilizes a unique thermal-cycling technology and fluorescence-based detection system to enable rapid and sensitive analysis of target sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Quick rna microprep kit, by Zymo Research (3 mentions)
Rnaiso plus purification kit, by Takara Bio (2 mentions)
Sybr green reaction reagent, by Takara Bio (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Iscript reverse transcription supermix, by Bio-Rad (2 mentions)
Itaq universal sybr green supermix, by Bio-Rad (2 mentions)
Aurum fatty and fibrous tissue kit, by Bio-Rad (2 mentions)
Lightcycler 480 sybr green 1 master mix, by Roche (1 mentions)
Diastar rt kit, by Solgent (1 mentions)
Sybr premix, by Takara Bio (1 mentions)
Moloney murine leukemia virus reverse transcriptase mml 5, by Promega (1 mentions)
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna plus kit, by Macherey-Nagel (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Pcr purification kit, by Qiagen (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
One step primescript mirna cdna synthesis kit, by Takara Bio (1 mentions)
15 protocols using lightcycler machine
1
Quantifying Renal and Pulmonary Gene Expression
2
Quantifying Brain Gene Expression
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Total RNA was extracted from brain tissue using the RNAiso Plus Purification Kit (9108; Takara Biotechnology Co., Ltd.). RNA was evaluated by A260 (OD) measurement and integrity was checked by 2% agarose gel electrophoresis. RNA was reverse transcribed into cDNA using PrimeScript™ RT Reagent Kit for gDNA Eraser Reverse Transcript Reagent Kit (RR047A; Takara Biotechnology Co., Ltd.). qPCR was performed in a LightCycler machine (Roche) with commercial SYBR-Green reaction reagent (RR820A; Takara Biotechnology Co., Ltd.). GAPDH was used as an internal control. The conditions for PCR consisted of 40 cycles of 95°C for 5 sec and 60°C for 20 sec followed by extension at 72°C for 10 min. The primer sequences used for gene amplification (Invitrogen; Thermo Fisher Scientific, Inc.) were as follows: MFSD2A-forward, 5′-CCACATTCACCATCCCTATCT-3′ and reverse, 5′-TTCTTATTCTGTCGCCGCTTC-3′, GAPDH-forward, 5′-ATGCCGCCTGGAAACC-3′ and reverse, 5′-GCATCAAAGTGGAAGAATGG-3′. The amplified products were 199 bp in length; gene expression was quantified via the 2−∆∆Cq method (20 (link)).
3
Quantitative RNA Expression Analysis
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Total RNA was isolated from tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized from 2 µg of total RNA using Moloney murine leukemia virus reverse transcriptase (MMLV) (Promega, Madison, WI, USA). The PCR products were analyzed by electrophoresis on 1.5% agarose gel. The electrophoretic products were photographed using a gel imaging system. Real-time quantitative PCR using SYBR Premix (DRR041A, TaKaRa, Japan) was performed according to the producer's specifications. Amplifications were performed in a LightCycler machine (Roche Diagnostics, Basel, Switzerland) following the manufacturer's instructions. A uniform amplification of the products was rechecked by analyzing the melting curves of the amplified products. 18s rRNA was used as an endogenous control for each sample. All samples were assayed in triplicate. The primer sequences used to amplify specific target genes were listed in Table 1 .
4
Quantification of mRNA Expression Levels
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The mRNA expression levels of ATP13A2-4 were quantified by RT-qPCR analysis. Therefore, we extracted RNA from 1.0 × 106 cells using the NucleoSpin RNA Plus Kit (740984, Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. The concentration and purity of the RNA samples were measured using a Nanodrop spectrophotometer (Thermo Fisher), followed by the conversion of RNA into cDNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (K1631, Thermo Fisher). The cDNA was then subjected to SYBR Green-based qPCR with gene-specific primer pairs (Table 1 ). β-actin was used as a reference gene. The analysis was performed using a Light Cycler machine (Roche) and the cycling conditions were as follows: 10 min at 95 °C, 50 cycles: 10 s at 95 °C, 30 s at 55 °C, 1 min at 95 °C and 1 min at 55 °C. Melting curves were analyzed from 55 to 95 °C. Finally, the mean Cq values were determined.
5
RNA Extraction and qRT-PCR Analysis
6
Chromatin Immunoprecipitation and Sequencing
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ChIP was performed as described using anti-HA 12CA5, anti-Myc 9E10 or anti-Pk(V5) antibody (Fernius et al., 2013 (link)). Primers used for qPCR analysis are given in Supplementary file 2B . qPCR was performed in a 20 μl Express SYBR GreenER (Life Technologies, Carlsbad, CA) reaction using a Lightcycler machine (Roche, Switzerland). To calculate ChIP enrichment/input, ΔCT was calculated according to: ΔCT = (CT(ChIP) − [CT(Input)− logE (Input dilution factor)]) where E represents the specific primer efficiency value. Enrichment/ input value was obtained from the following formula: E^−ΔCT. qPCR was performed in triplicate, typically for each of three or more independent experimental repeats. Error bars represent standard error. For ChIP-Seq, purified chromatin was recovered using a PCR purification kit (Qiagen, Netherlands) followed by drying in a speedvac. Samples were sequenced on a HiSeq2000 instrument (Illumina, San Diego, CA) by the EMBL Core Genomics Facility (Heidelberg, Germany). The summary of data obtained is given in Supplementary file 2C .
7
Detecting miR-148b and MTA2 Expression
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Total RNA of cells or tissues was extracted using TRIzol reagent (Thermo Fisher Scientific). The cDNA was synthesized using the PrimeScript RT Reagent Kit (Takara, Dalian, China). For the detection of miR-148b level, miRNAs were reversely transcribed using the One Step PrimeScript miRNA cDNA Synthesis Kit (Takara) and the amplification processes were performed using the SYBR Premix Ex Taq II Kit (Takara) by a Light Cycler machine (Hoffman-La Roche Ltd, Basel, Switzerland). The primers for miR-148b and U6 small nuclear RNA (snRNA) were purchased from Ambion (Thermo Fisher Scientific). U6 snRNA was used as an internal control of miR-148b. For the detection of MTA2 mRNA level, the cDNA was synthesized using the Prime-Script RT Reagent Kit (Takara) and then amplified using an SYBR Green PCR Kit (Takara). The primers for MTA2 and β-actin were described previously,13 (link) and β-actin was used as an internal control of MTA2. The PCR results were normalized to internal controls and analyzed according to the 2−ΔΔCt method. Three independent experiments were performed.
8
RNA Extraction and Real-time PCR Analysis
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An RNAiso Plus Purification Kit (TaKaRa Biotechnology Co., Ltd, 9108) was used to extract total RNA from the cardiomyocytes. The RNA concentration was evaluated based on the optical density of the sample at 260 nm, and the RNA integrity was assessed through 2% agarose gel electrophoresis. The RNA was reverse-transcribed into cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa Biotechnology Co., Ltd, RR047A) [71 (link), 72 (link)]. Real-time PCR was performed on a LightCycler machine (Roche) with a commercial SYBR Green reaction reagent (TaKaRa Biotechnology Co., Ltd, RR820A). GAPDH was used as an internal control. The cDNA was denatured for 30 s at 95 °C, followed by 40 cycles of 5 s at 95°C [73 (link)].
9
Cardiac Tissue RNA Extraction and qPCR
10
RNA Extraction and qRT-PCR Analysis
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Total cellular RNA was extracted from homogenized tissues and sorted cells using TRIZOL reagent (Invitrogen) following the manufacturer's protocol. cDNA was prepared using cDNA archive kit (Applied Biosystems), and quantitative RT-PCR was performed using the Light Cycler-FastStart DNA Master SYBR Green I kit (Roche Diagnostics) on a LightCycler machine (Roche Diagnostics). mRNA expression was measured using the QuantiTect SYBR Green PCR primers (Qiagen).
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