Total RNA was extracted from 100 mg of A. thaliana leaves using the RNeasy Plant Mini Kit RNA from Qiagen (Germantown, MD). Two micrograms of RNA were used for retro-transcription with FastGene Scriptase II cDNA-Kit (Nippon Genetics Europe, Düren, Germany). qPCR was performed in triplicates by using specific qPCR primers (Sigma-Aldrich Corporation, St. Louis, MO), the qPCRBIO SyGreen Mix (PCR Biosystem, London, UK) and the Real-Time PCR Light-Cycler II (Roche Diagnostics, Indianapolis, IN). mRNA levels were normalized to actin (ACT8), GAPDH and tubulin beta chain (TUB8) transcripts, and the relative mRNA levels were determined by using the 2-ΔΔCt method (Pfaffl, 2001 (link)). The primer sequences are listed in Supplemental Table S1
Qpcr primers
Manufactured by Merck Group
Sourced in United States
QPCR primers are short DNA sequences used in quantitative polymerase chain reaction (qPCR) experiments. They are designed to specifically bind to and amplify target DNA sequences, enabling the quantification of gene expression or the detection of specific DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Real time pcr lightcycler 2, by Roche (2 mentions)
Rna rneasy plant mini kit, by Qiagen (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Fastgene scriptase 2 cdna kit, by Nippon Genetics (1 mentions)
Ab211091, by Abcam (1 mentions)
Powerwave xs, by Agilent Technologies (1 mentions)
D8418, by Merck Group (1 mentions)
Nucleospin rna extraction kit, by Macherey-Nagel (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
Icycler iq real time pcr detection system, by Bio-Rad (1 mentions)
Taqman preamp master mix kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Lightcycler 480 sybr green 1 master mix, by Roche (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Anti lc3, by Novus Biologicals (1 mentions)
8 protocols using qpcr primers
1
Quantitative Gene Expression Analysis in Arabidopsis
2
Evaluation of ABCE1 and TET2 Knockdown on Cell Viability
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Levels of ABCE1 mRNAs were reduced by reverse transfection with 27-mer duplex siRNAs targeting ABCE1. Increasing concentrations (0, 2.5, 5, and 10 nM) of scrambled (scr, SR30004; OriGene) or ABCE1 siRNAs diluted in Opti-MEM (31985070; Thermo Fisher Scientific) were transfected in triplicates in 48-well plates with Lipofectamine RNAiMAX (13778075; Thermo Fisher Scientific) before seeding EV, TET2 CD, and TET2 mCD cells (2.5 × 104 in each well). After 48 h, cell viability was quantified using an MTT detection kit (ab211091; Abcam). The medium was replaced by 100 μl of a 1:1 mix of phenol red–free DMEM without serum and a 10% MTT solution, and the plate was further incubated for 3 h at 37°C. The formed insoluble formazan crystals were then dissolved with a 1:1 solution of DMSO (D8418; Sigma-Aldrich) and isopropanol (33539; Sigma-Aldrich). After 30 min of incubation and a transfer into a 96-well plate, absorbance was detected at OD = 570 nm using a BioTek microplate reader (Power wave XS). For TET knockdown, 10 nM of siRNAs were reverse-transfected in MCF-7, T47D, and HEK293T cells as described earlier. After 48 h, total RNAs were extracted and subjected to RT-qPCR analysis. All siRNA sequences are listed in Table S2.
Table S2
3
qPCR Primer Synthesis and Characterization
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The qPCR primers were synthesized by Sigma-Aldrich (USA) and the list of primers used in this study is provided in S1 and S2 Tables.
4
Quantifying Gene Expression in Macrophages
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The total RNA was isolated from macrophages (infected and uninfected) using NucleoSpin RNA extraction kit (Macherey-Nagel, Germany) according to the manufacturer's protocol. RNA (1 μg) was converted to cDNA using the GoScript reverse transcription system (Promega, Madison, WI). The cDNA synthesized was used for qPCR using the iQ SYBR green SuperMix (Bio-Rad Laboratories, Hercules, CA) on an iCycler iQ real-time PCR detection system (Bio-Rad, USA). Fold changes in gene expression levels were calculated and were normalized to human actin. The list of quantitative real-time PCR (qPCR) primers (from Sigma-Aldrich, USA) is shown in Table S2.
5
Quantitative PCR Expression Analysis
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Quantitative PCR (qPCR) primers (Sigma) were designed using Primer-BLAST (NCBI; sequences are detailed in Supplementary Table 5 ). A total of 10 ng of cDNA was used for quantitative PCR in a total volume of 10 µl with LightCycler 480 SYBR Green I Master Mix (Roche, Switzerland) and specific primers (detailed in Supplementary Table 5 ), on a LightCycler 480 (Roche, Switzerland). The amplification conditions were determined by the primers to present amplification efficiency close to 100% and a single peak in melt-curve analyses. In sorted cell samples, genes of interest were pre-amplified by TaqMan™ PreAmp Master Mix kit (Applied Biosystems) following manufacturer´s instructions. Each Real-time PCR reaction was performed in triplicates. Glyceraldehyde 3‐phospate dehydrogenase (GAPDH) and Actb (β-actin) were used as housekeeping gene. The log fold change in mRNA expression was calculated from ΔΔCt values relative to control samples.
6
RNA Extraction and qPCR Analysis Protocol
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RNA was extracted from cells or tissues using TRIzol (Thermo Fisher Scientific) extraction according to the manufacturer’s instructions. Briefly, cells and tissues were resuspended in 1 ml TRIzol reagent and lysed using a tissue lyser. Cells were then mixed with 200 µl chloroform and incubated for 15 min at RT. Cells were spun down at 12,000 rpm for 15 min, and the aqueous phase was transferred to a new tube. Isopropanol was added to precipitate RNA and spun down. The RNA pellet was washed twice with 75% EtOH, and the pellet was resuspended in distilled H2O. cDNA was transcribed from isolated RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. Real-time PCR was performed using the SYBR Select Master Mix (Applied Biosystems) according to the manufacturer’s instructions. qPCR primers were purchased from Sigma-Aldrich. To analyze relative mRNA levels, the derived values were normalized to 18s mRNA levels.
7
LRRK2 Kinase Inhibitor-Mediated Cellular Assays
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LRRK2 kinase inhibitor-1 (L2-IN1), HG 10-102-01 and CZC-25146 were purchased from Calbiochem, GSK2578215A from Tocris and PF-06447475, MG132 and chloroquine from Sigma-Aldrich. MLi-2 was kindly provided by Dr. D. Alessi (Division of Signal Transduction Therapy, University of Dundee). Antibodies used are as follows: anti-flagM2, anti-vinculin and anti-α or β-tubulin (Sigma-Aldrich), anti-LRRK2 MJFF-2, anti-LRRK2 P-S935, anti-LC3 (Novus Biologicals) and anti-GAPDH (Abcam). QPCR probes were from Sigma-Aldrich, QPCR primers from IDT. Lentiviral vectors encoding full length LRRK2 were produced by the Leuven viral vector core (https://gbiomed.kuleuven.be/english/research/50000715/laboratory-of-molecular - virology-and-gene-therapy/lvvc).
8
Quantifying Arabidopsis AHA1 and AHA2 Transcripts
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Total RNA was extracted from 100 mg of Arabidopsis leaves using the RNeasy Plant Mini Kit RNA from Qiagen (Hilden, GE). Two micrograms of RNA were used for retro-transcription with M-MLV from Invitrogen (Carlsbad, CA). qPCR was performed in triplicates by using validated qPCR primers from Sigma (St. Louis, MO) specific for AHA1 and AHA2 transcripts, KAPA SYBR FAST qPCR Master mix from KapaBiosystems (Boston, MA) and the Real-Time PCR Light-Cycler II from Roche Diagnostics (Indianapolis, IN). mRNA levels were normalized to beta 8-tubulin mRNA (TUB8), and the relative mRNA levels were determined by using the 2 ÀDDCt method (Pfaffl, 2001) .
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