Total RNAs, including the miRNAs, were isolated from the skeletal muscle biopsies (approximately 30 mg) of 12 patients representing four different MDs and 3 control subjects using the miRCURY RNA Isolation Kit-Cell & Plant (Qiagen, Valencia, CA), according to the manufacturer's instructions. RNA integrity and concentration were assessed using the agarose gel electrophoresis and NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA), respectively.
Mircury rna isolation kit cell plant
Manufactured by Qiagen
Sourced in United States, Denmark
The MiRCURY™ RNA Isolation Kit-Cell & Plant is a lab equipment product designed for the isolation and purification of total RNA, including small RNAs, from a variety of cell and plant samples.
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Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Exo check exosome antibody array, by System Biosciences (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Prism v7, by GraphPad (1 mentions)
Dynamo colorflash sybr green, by Roche (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Primescript reverse transcription kit, by Takara Bio (1 mentions)
Agilent small rna kit, by Agilent Technologies (1 mentions)
Agilent rna 6000 pico kit, by Agilent Technologies (1 mentions)
Mircury rna isolation kit biofluid, by Qiagen (1 mentions)
Quant it ribogreen, by Thermo Fisher Scientific (1 mentions)
Agilent rna 6000 pico kit and small rna kit, by Agilent Technologies (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
20 protocols using mircury rna isolation kit cell plant
1
RNA Isolation from Skeletal Muscle
2
Quantification of miRNA and mRNA Levels
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RNA was extracted using the miRCURY™ RNA Isolation Kit – Cell & Plant (QIAGEN, Hilden, Germany) and miScript II RT Kit (QIAGEN) following the manufacturer's protocols. For cDNA synthesis 1 μg of RNA was used. Real-time PCR amplification and analysis was performed using a LightCycler 480 with DyNAmo ColorFlash SYBR Green (Roche, Mannheim, Germany) and the primers listed in Supplementary Table S2 . For normalization, HPRT1, YWHAZ and RPS29 mRNAs were used as controls. miRNA quantification was performed by using the miRNA miScript Primer assay and miScript SYBR Green PCR Kit (QIAGEN). U6, SNORD61 and SNORD95 (QIAGEN) were used as controls. Calculations of relative changes in gene expression were done using the ΔΔ cycle threshold (Ct) method (25 (link)). For statistical analysis the unpaired two sample Student's t-test was applied, using the software GraphPad prism V 7.03 (GraphPad Software, San Diego, USA).
3
RNA Isolation and RT-qPCR Analysis
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RNA from PBMCs was isolated with miRCURYTM RNA Isolation Kit-Cell & Plant (Exiqon, Aarhus, Denmark) following manufacturer’s instructions. RNA was quantified with a NanoDrop 2000 and integrity was assessed in a 2% agarose gel. A total of 500 µL of RNA were retro-transcribed with PrimeScript Reverse Transcription Kit (Takara, Saint-Germain-en-Laye, France) according to manufacturer’s protocol and resulting cDNA was used as a template for amplifying MDM2 and DENND5B genes by RT-qPCR with FastStart Universal SYBR Green Master. Primers used for MDM2 amplification were 5′-CCCAAGACAAAGAAGAGAGTGTGG-3′ and 5′-CTGGGCAGGGCTTATTCCTTTTCT-3′ forward and reverse primers, respectively. To amplify DENND5B, we used 5′-CTCCAGCGATACAACTCCTATGA-3′ and 5′-GTGGATATAGCTTTCAAGTGGCA-3′ forward and reverse primes, respectively. Relative gene expression was calculated with the 2−ΔΔCt method using the quadratic average of RN18S and RPLP0 CTs as references.
4
Total RNA Extraction and Analysis
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Total RNA was extracted using the miRCURYTM RNA Isolation Kit - Cell & Plant (Exiqon, Vedbæk, Denmark) according to the manufacturer′s protocol.
The concentration of isolated total RNA was determined using a NanoDrop Lite spectrophotometer (Thermo Scientific, Hudson, NH, USA). The total RNA profiles were
visualized by using an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and Agilent RNA6000 Pico Kit (Agilent Technologies). In addition, the
miRNA (10–40 nt) concentrations were analyzed by using an Agilent 2100 bioanalyzer (Agilent Technologies) and Agilent Small RNA Kit (Agilent Technologies)
according to the manufacturer′s protocols.
The concentration of isolated total RNA was determined using a NanoDrop Lite spectrophotometer (Thermo Scientific, Hudson, NH, USA). The total RNA profiles were
visualized by using an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and Agilent RNA6000 Pico Kit (Agilent Technologies). In addition, the
miRNA (10–40 nt) concentrations were analyzed by using an Agilent 2100 bioanalyzer (Agilent Technologies) and Agilent Small RNA Kit (Agilent Technologies)
according to the manufacturer′s protocols.
5
Plasma and Macrophage MicroRNA Extraction
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MicroRNA-enriched total RNA from plasma and macrophages was isolated with miRCURY™ RNA Isolation Kit-Biofluids (Exiqon, Madrid, Spain) and miRCURY™ RNA Isolation Kit-Cell & Plant (Exiqon, Madrid, Spain), respectively. The extracted RNA was quantified using NanoDrop 2000 (Isogen LifeSciences, Utrecht, Netherlands).
6
Exosomal RNA Extraction and Analysis
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EV RNA extraction was performed using the miRCURY RNA Isolation Kit - Cell & Plant (#300110, Exiqon) following the manufacturer’s instructions. In brief, exosome pellet was lysed with the provided lysis solution supplemented with 96–100% ethanol, and the mixture was loaded to the column. The EV RNA was washed and eluted with 15–50 µL of RNase-free water. The extracted RNA concentration was calculated by Quant-IT RiboGreen (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA). RNA size was confirmed using Agilent RNA 6000 Pico Kit and Small RNA Kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany).
7
Profiling miRNAs in sEVs from Antiphospholipid Syndrome
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miRNAs contained in sEVs from aPL, PAPS and HD (n = 8) were extracted following the instructions of miRCURY RNA Isolation Kit—Cell & Plant (Exiqon, Woburn, MA, USA). miRNA samples were processed and sequenced (single end, 50 nts, 1 × 50, v4) by CNAG (Barcelona, Spain) on a HiSeq2500 Illumina device with a read depth of >10 M reads/sample. Subsequent quality control, data processing and analysis were performed as previously described [48 (link),49 (link),50 (link)]. Data were deposited in the Gene Expression Omnibus (NCBI) with the number GSE220791 (more details in Supplementary Materials ).
8
Quantification of Cellular mRNA Expression
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Total RNA was isolated from cell lines and exosomes using the miRCURY RNA Isolation Kit – Cell & Plant (Exiqon, Woburn, MA) followed with spectrophotometry (NanoDrop, Thermo Scientific) for quantification and qualitative analysis. Equal amounts of total RNA was reverse-transcribed by oligo (dT) priming using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA) following the manufacturer's instructions. Real-time PCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) and the SYBR Green PCR Master Mix (Applied Biosystems) as described previously [40] (link). Glut1 and β-Actin primers were purchased from SABiosciences (Frederick, MD).
9
Urinary RNA Extraction and Analysis
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RNA from urinary pellets or exosomes were extracted using the miRCURY™ RNA Isolation Kit-Cell & Plant (Exiqon, Woburn, MA, USA, more details in SI ), following the manufacturer’s instructions. Quantification and evaluation of the RNA quality was assessed by Bioanalyzer PicoChip analysis.
10
Extracting RNA from Urinary Extracellular Vesicles
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To extract RNA from uEVs isolated by ultracentrifugation, we employed miRCURY™ RNA Isolation Kit Cell & Plant (Exiqon). In average, 1.5e7 vesicles were used per retrotranscription reaction. In addition, a set of samples was extracted by Norgen Biotek Exosomal RNA purification kit, following the manufacturers' instructions. For cell lines, RNA was extracted using NucleoSpin® RNA isolation kit from Macherey-Nagel (ref: 740955.240C). cDNA was synthesized from 0.1–1 μg of RNA using Superscript III (Life Technologies) following the manufacturer's recommendations. For prostate tissue samples, RNA was extracted as reported in [16 ]. Quantitative Real Time PCR (Taqman qRTPCR) was performed as previously described [18 (link)]. Universal Probe Library (Roche) primers and probes employed are detailed in Supplementary Table S2 . β-ACTIN (Hs99999903_m1) and GAPDH (Hs02758991_g1) housekeeping assays were from Applied Biosystems and showed similar results.
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