THP-1 cells were collected by centrifugation at 1200 rpm for 5 min and the supernatant was transferred to 1.5 mL tubes (Eppendorf, Hamburg, Germany). Cells were lysed with lysis buffer (50 mL Pierce M-PER Mammalian Protein Extraction Reagent, 1 tablet of Roche Protease Inhibitor Cocktail, 10 mM Lactose Monohydrate) or homogenized with 200 µL of homogenization solution from the Maxwell® 16 LEV simplyRNA Cells kit (Promega, Madison, WI, USA). Total RNA was extracted using Maxwell® 16 LEV simplyRNA Cells kit in a Promega Maxwell® 16 Instrument according to the manufacturer’s instructions. RNA samples were stored at −80 °C.
Maxwell 16 lev simplyrna cells kit
Manufactured by Promega
Sourced in United States
The Maxwell 16 LEV simplyRNA Cells Kit is a laboratory instrument designed for the automated purification of RNA from cells. The kit utilizes magnetic bead-based technology to extract and purify RNA samples in a streamlined, efficient manner.
Automatically generated - may contain errors
Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (5 mentions)
Maxwell 16 instrument, by Promega (4 mentions)
7900ht sequence detection system, by Thermo Fisher Scientific (3 mentions)
Abi 7900, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Promega (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Bioanalyzer rna 6000 nano kit, by Agilent Technologies (2 mentions)
Maxwell nucleic acid extraction instrument, by Promega (2 mentions)
Maxwell 16, by Promega (2 mentions)
1.5 ml tube, by Eppendorf (1 mentions)
M per mammalian protein extraction reagent, by Roche (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Truseq stranded total rna library prep kit, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Truseq stranded mrna sample prep kit, by Illumina (1 mentions)
Maxwell 16 lev instrument, by Promega (1 mentions)
Epmotion 5075 robot, by Eppendorf (1 mentions)
Sybr green technique, by Promega (1 mentions)
33 protocols using maxwell 16 lev simplyrna cells kit
1
THP-1 Cell Lysis and RNA Extraction
2
Comprehensive CircRNA Profiling in Human Cell Lines
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RNA was extracted using the Maxwell 16 LEV simplyRNA Cells Kit (Promega) from SH-SY5Y (human neuroblastoma) and Jurkat E6-1 (human T lymphocyte) cell lines. RNA quality was assessed by the LabChip GX Touch instrument (PerkinElmer, Waltham, MA, United States). RNA sequencing was performed using the TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA, United States), following the manufacturer’s instructions and a paired-end sequencing strategy. SH-SY5Y and Jurkat samples underwent a high-coverage paired-end 75- and 150-bp strand-specific sequencing, respectively, using a NextSeq 500 platform (Illumina).
The circRNA analysis was then performed using the DCC software (Cheng et al., 2016 (link)). In detail, raw reads were first aligned to the hg19 version of the genome using STAR (Dobin et al., 2013 (link)), switching on the detection of chimeric alignments to detect reads containing backspliced products, as suggested by the DCC manual. In a first step, reads were mapped using both mates; subsequently, an additional separate mate mapping was performed. After mapping, DCC was used to analyze the chimeric reads to detect circRNAs. Only those circRNAs supported by at least five reads were considered for further analyses. CircRNAs mapping on mitochondrial DNA or in repetitive regions of the genome were filtered out.
The circRNA analysis was then performed using the DCC software (Cheng et al., 2016 (link)). In detail, raw reads were first aligned to the hg19 version of the genome using STAR (Dobin et al., 2013 (link)), switching on the detection of chimeric alignments to detect reads containing backspliced products, as suggested by the DCC manual. In a first step, reads were mapped using both mates; subsequently, an additional separate mate mapping was performed. After mapping, DCC was used to analyze the chimeric reads to detect circRNAs. Only those circRNAs supported by at least five reads were considered for further analyses. CircRNAs mapping on mitochondrial DNA or in repetitive regions of the genome were filtered out.
3
Cycloheximide-induced RNA Sequencing
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Five million cells were treated with cycloheximide (CHX, 100 μg/ml) for 5 min, followed by RNA isolation using the Maxwell 16 LEV simplyRNA Cells Kit (Promega). Sequencing libraries were generated using the TruSeq Stranded mRNA Sample Prep Kit (Illumina) and were sequenced on an Illumina HiSeq 2500 instrument.
4
Pneumococcal Gene Expression Analysis
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For gene expression analysis, pneumococcal strains were grown to an OD590 of 0.15 to 0.18 in triplicate. Five milliliters of cells was added to 1 ml of an ice-cold 95% ethanol–5% phenol solution, before centrifugation at 4,000 rpm for 10 min. The supernatant was removed, and the pellets were stored at −80°C until processing. RNA was extracted using the Maxwell 16 LEV simplyRNA cells kit (Promega, USA) and the Maxwell 16 LEV instrument (Promega, USA). The manufacturer’s protocol was followed from step 4, and the manufacturer’s lysis steps (steps 1 to 3) were replaced with the following protocol to improve cell lysis: pellets were resuspended in 50 μl TE with 3 mg/ml lysozyme and incubated at 37°C for 10 to 20 min. RNAseq analysis was done by using a MiSeq desktop processor (Illumina, USA) and the ScriptSeq complete kit (bacteria) (CamBio, UK), which includes an rRNA depletion step. Raw data were trimmed using Trimmomatic-0.32 and aligned using BWA-Mem and samtools. Expression data were generated using Rockhopper v2.0.3 (44 (link)) with D39 as the reference genome (GenBank accession number NC_008533.2 ).
5
Quantitative Gene Expression Analysis
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Gene expression was measured as already described [15 (link)]. Briefly, tumor samples, with more than 80% of tumor cells, were homogenized in RNA lysis buffer in ice with an Ultra-Turrax (IKA, Staufen, Germany), and RNA was purified using the Maxwell 16 LEV SimplyRNA Cells kit (Promega, Madison, WI, USA). In all the samples by real time-PCR, the % of murine DNA contamination was established using primers specifically designed to distinguish human from murine actin and only samples with more than 85% human DNA were processed. Retro-transcription to cDNA was done using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). Genes selected were DNA pol β, ERCC1 and XPF. Optimal primer pairs (Table S3 ) were chosen, spanning splice junctions, using Primer3 Input software (Primer3 Input. http://primer3.ut.ee/ ) and the human specificity was verified by detecting single-band amplicons of the PCR products. Absolute copy numbers of mRNA were determined by real time-PCR (ABI-7900, Applied Biosystems, Foster City, CA, USA) with the SYBR Green technique (Promega), using an EP Motion 5075 robot (Eppendorf, Hamburg, Germany). Standard curves for each gene were included for absolute quantification of mRNA.
Real time-PCR data were normalized using the geometric mean of two housekeeping genes, actin (ACTB) and ciclophillin (CYPA).
Real time-PCR data were normalized using the geometric mean of two housekeeping genes, actin (ACTB) and ciclophillin (CYPA).
6
RNA-seq Analysis of E. faecalis Variants
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RNA-seq analysis in E. faecalis H25 variants was carried out as described previously (23 (link)). Bacteria were grown to mid-log phase in BHI broth, harvested by centrifugation and lysed with 10 μg/ml lysozyme (Sigma, UK) and 10 U/ml Mutanolysin (Sigma, UK). RNA was extracted using the Maxwell® 16 LEV SimplyRNA Cells Kit (Promega, USA) following the manufacturer's instructions. After depletion of rRNAs with Ribo-Zero from Illumina, the RNA samples were sequenced as 75-bp paired-end reads on an Illumina HiSeq 4000 and trimmed with trimmomatic/0.32 (44 (link)). Trimmed FASTq reads were aligned to the E. faecalis H25 genome generated by SMRT sequencing (accession GCA_002289045.2). RNA-seq was performed in triplicates on one clone for each variant and then a single RNA-seq for the second clone of each variant. The FASTq reads have been deposited in the SRA (accession PRJNA400682).
7
Quantitative Gene Expression Analysis
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RNA was extracted from frozen liver samples using the Maxwell 16 LEV simplyRNA Cells Kit (Promega) following the manufacturer’s recommendations. Two micrograms of RNA, treated with DNase I, was retro-transcribed using Moloney Murine Leukemia Virus (M-MLV) retro-transcriptase (Invitrogen) and random primers (Life Technologies, Thermo). cDNA was amplified, and relative gene expression was determined by qPCR using iQTM SYBR Green Supermix reagent (Bio-Rad) in CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Table 1 contains the sequence of primers specific for the transgene (CYP27A1) and the mouse genes Cyp7a1, Cyp3a11, Cyp27a1 (exons 1/2), Cyp27a1 (exons 8/9), and 36b4 (used as a housekeeping gene).
Delta cycle threshold (ΔCt) values using 36b4 mRNA levels as reference gene were corrected with the efficiency of amplification of each pair of primers and multiplied by 1,000 to facilitate graphical representation.
Delta cycle threshold (ΔCt) values using 36b4 mRNA levels as reference gene were corrected with the efficiency of amplification of each pair of primers and multiplied by 1,000 to facilitate graphical representation.
8
RNA Decay Kinetics in HeLa Cells
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Hela cells (control, TIAR and ELAVL1 knockdown) were seeded in 12-wells plate at a density of 1.6 × 105 cells per well. The general transcription inhibitor actinomycin D (Sigma Aldrich) was added at a concentration of 5 g/ml for 0, 4, 8 and 12 h. Total RNA was isolated with Maxwell 16 LEV simply RNA cells kit (Promega) and analyzed by RT-qPCR. ACTB was used as a reference gene for normalization (ΔCt), and the ΔCt of each time point was compared to time 0 h (ΔΔCt). For each experiment the average of triplicate wells per condition was calculated and used to determine mean values and standard deviation of three biological replicates. A Student's t-test was performed to assess for significant differences of RNA decay rate between the three conditions.
9
MDR-1 and c-Met Expression Assay
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RNA was extracted by using Maxwell 16LEV simplyRNA Cells kit (Promega). RNA was retro-transcribed to cDNA using High Capacity cDNA Reverse Transcription Kit (Life Technologies). Differences in MDR-1 gene expression were determined by real-time RT-PCR performed with Sybr Green PCR master mix (Promega) and the dissociation curve was evaluated for each gene. Samples were then normalized using the expression of the housekeeping gene (actin) and their levels were compared to control samples. Real-time PCR was done using the 7900HT Sequence Detection System (Applied Biosystems).
The c-Met gene copy number was assessed using the TaqMan Copy Number Assay (Applied Biosystems) with the ABI 7900, Applied Biosystems. hTERT copy number was used as reference gene.
The c-Met gene copy number was assessed using the TaqMan Copy Number Assay (Applied Biosystems) with the ABI 7900, Applied Biosystems. hTERT copy number was used as reference gene.
10
RNA Isolation from PBMC Samples
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