Mirvana paris total rna isolation kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The MirVana PARIS total RNA isolation kit is a product designed for the purification of total RNA from various sample types. It utilizes a proprietary methodology to efficiently isolate both large and small RNA molecules, including microRNAs. The kit provides a straightforward and reproducible approach to total RNA extraction.

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4 protocols using mirvana paris total rna isolation kit

Validating miRNA Expression via qPCR

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To confirm the results from miRNA-Seq, we selected some miRNAs of interest to measure using PCR. Total RNA was isolated from AMSCs by the mirVana PARIS total RNA isolation kit (Life Technologies, Carlsbad, CA, USA, Cat# AM1556) according to the kit protocol. RNA concentrations were measured by a NanoDrop Spectrophotometer (NanoDrop, Thermo Fisher Scientific, Inc.). A fixed volume of 5 μL of RNA elute at 1 ng/uL was reverse transcribed by using the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies, Cat# 4366596). For PCR, 1.33 μL of RT product was combined with 10 μL of TaqMan Universal Master Mix (Cat# 4440038), 7.67 μL of H2O and 1 μL of primers, including miR-21, miR-146a, miR-155, and miR-210 (Life Technologies, Cat# 000397, 001097, 002623, and 000512 respectively) to make up a 20 μL reaction. RNU6B (Life Technology Cat# 001093) was included in the assay as reference control. Real-time PCR was carried out on an Applied Biosystems (Foster City, CA, USA) ViiA7 Real-Time PCR system at 50 °C for 2 min, 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Fold changes of miRNA levels in hypoxia relative to normoxia were calculated using the 2^−ΔΔCt method.

Saad A., Zhu X.Y., Herrmann S., Hickson L., Tang H., Dietz A.B., van Wijnen A.J., Lerman L, & Textor S. (2016). Adipose-derived mesenchymal stem cells from patients with atherosclerotic renovascular disease have increased DNA damage and reduced angiogenesis that can be modified by hypoxia. Stem Cell Research & Therapy, 7(1), 128.

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mRNA Sequencing and Proteomic Analysis of MSC-derived EVs

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mRNA was isolated from MSC-derived EVs using the mirVana PARIS total RNA isolation kit (Life Technologies) according to the manufacturer’s protocol. mRNA sequencing was performed at the Mayo Clinic Bioinformatic Core, as previously described^{13 (link)}. Samples were sequenced on an Illumina HiSeq 2000 using TruSeq SBS kit version 3 and HCS v2.0.12 data collection software and data analyzed using the MAPRSeq v.1.2.1 system and the Bioinformatics Core standard tool, which includes alignment with TopHat 2.0.6^{27 (link),28 (link)} and gene counts with the featureCounts software^{29 (link)}. Normalized expression values for each gene were calculated as reads per kilobase per million (RPKM).
In addition, liquid chromatography mass spectrometry (LC-MS/MS) proteomic analysis was performed as previously described^{30 (link),31 (link)}. EV pellets were solubilized and lysed, and protein samples denatured. Aliquots were resolubilized in reducing sample buffer and samples electrophoresed. Gel sections were digested with trypsin^{31 (link)}, and peptides extracted and transferred onto a PicoFrit column 9 (NewObjective), self-packed with Agilent Poroshell 120 S 2.7 µm EC-C18 stationary phase, using a Dionex UltiMate 3000 RSLC LC system (Thermo-Fisher Scientific). Peptides were separated and eluting peptides analyzed using a QExactive mass spectrometer (Thermo-Fisher Scientific).

Eirin A., Zhu X.Y., Jonnada S., Lerman A., van Wijnen A.J, & Lerman L.O. (2018). Mesenchymal Stem Cell-Derived Extracellular Vesicles Improve the Renal Microvasculature in Metabolic Renovascular Disease in Swine. Cell Transplantation, 27(7), 1080-1095.

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miRNA and mRNA Profiling in Myocardium

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Cells were washed once in PBS and pelleted at 1000 rpm for 5 minutes before being re-suspended in 200μL of Cell Disruption Buffer from the miRvana Paris Total RNA Isolation Kit (LifeTechnologies). RNA isolation was performed as per the manufacturer’s protocol. RT and qPCR for miRNA and mRNA quantification was performed in an identical manner as described above. We measured myocardial expression of selected miRNAs among those in the second PC with a loading greater than 0.60 (see Figure 1), including miR-200a-3p, miR-208a-3p, miR-212-5p, miR-423-5p, miR-193b-5p, miR-15a-5p, miR-221-5p). We next selected joint mRNA targets as those mRNAs targeted by 3 miRNAs in the network (14 mRNAs). A list of genes, miRNAs, and known function is shown in Supplemental Table 1. For cellular expression experiments (fibroblasts, endothelial cells, neonatal rat ventricular myocytes), RNA was isolated from cells using Trizol according to the manufacturer’s protocol. Quantitative RT-qPCR for miRNA expression was performed with 80 ng RNA using the miRCURY LNA Universal RT microRNA PCR kit from Exiqon per the manufacturer’s protocol (with normalization to a spike-in UniSp6).

Shah R., Ziegler O., Yeri A., Liu X., Murthy V., Rabideau D., Xiao C.Y., Hanspers K., Belcher A., Tackett M., Rosenzweig A., Pico A.R., Januzzi J.L, & Das S. (2018). MicroRNAs Associated With Reverse Left Ventricular Remodeling in Humans Identify Pathways of Heart Failure Progression. Circulation. Heart failure, 11(2), e004278.

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Quantifying Immune Markers in Adipose Tissue

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Frozen abdominal or pericardial fat tissues (~50mg) were homogenized in 350ul of ice cold lysis buffer, supplied by mirVana PARIS total RNA isolation kit (Life Technologies, Cat# AM1556).
Total RNA was then isolated from homogenized samples, and its concentration measured by a Spectrophotometer (NanoDrop). 50μl of the RNA samples were then treated with 2 units for rDNase (Life Technologies, Cat# AM1906) to remove possible contaminating DNA. First-strand cDNA was produced from 300ng of total RNA using SuperScript VILO cDNA Synthesis kit (Life Technologies, Cat#11754-050). Relative quantitative PCR was performed using Taqman assays, containing 20ng of cDNA products. Taqman assays included the followings: iNOS (Ss03374608), CD86 (Ss03394398), mannose-receptor (Ss03373693), CD11c (AJ1RVEU), TNF-a (Ss03391318), IL-6 (Ss03384604), CD-68 (AJWR2PY), arginase-1 (Ss03391398) and GAPDH (Ss03374854, for internal control), all from Life Technologies. Negative controls with no cDNA were cycled in parallel with each run. PCR analysis was done on Applied Biosystems ViiA7 Real-Time PCR systems at the following conditions: 50°C for 2 minutes, 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Fold-changes of each target gene in the obese relative to lean group were calculated using the 2^-ΔΔCT method. Fold changes greater than 2 are considered as significant.

Pawar A.S., Zhu X.Y., Eirin A., Tang H., Jordan K.L., Woollard J.R., Lerman A, & Lerman L.O. (2014). Adipose tissue remodeling in a novel domestic porcine model of diet-induced obesity. Obesity (Silver Spring, Md.), 23(2), 399-407.

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