Mirvana paris rna and native protein purification kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The MirVana™ PARIS™ RNA and Native Protein Purification Kit is a laboratory tool designed to isolate and purify both RNA and native proteins from the same sample. It provides a streamlined method for the simultaneous extraction and preservation of these biomolecules.

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MirVana PARIS RNA purification kit MirVanaTM PARISTM RNA kit MirVana RNA purification kit MirVana PARIS Kit MirVana PARIS RNA purification kit PARIS™ Kit MirVana kit

23 protocols using mirvana paris rna and native protein purification kit

Spinal Cord Protein Quantification

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The L5 dorsal spinal cord was homogenized with Cell Disruption Buffer in mirVana PARIS RNA and Native Protein Purification Kit (Thermo Fisher Scientific). Lysates were centrifuged at 12,000 rpm for 20 min at 4°C and the supernatants were collected. ELISA was performed using RayBio ELISA kits for rat CCL2, IL-1β, and IL-6 (RayBiotech, Norcross, GA) according to the manufacturer’s protocols.

Maruyama M., Sakai A., Fukunaga T., Miyagawa Y., Okada T., Hamada M, & Suzuki H. (2023). Neat1 lncRNA organizes the inflammatory gene expressions in the dorsal root ganglion in neuropathic pain caused by nerve injury. Frontiers in Immunology, 14, 1185322.

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Corneal Tissue Microdissection and Analysis

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Specific microdissection was performed to provide portions suitable for 30-µm radial or tangential cross-sectioning. Briefly, corneoscleral specimens were dissected into three portions: four limbal, four corneal, and four conjunctival squares, all belonging to the same corneoscleral specimen. Cutting activity was carried out under a dissecting stereomicroscope (SMZ645; Nikon) equipped with cold-light optic fibers (PL2000 photonic; Axon, Vienna, Austria), under the supervision of an expert pathologist. Limbal zone was defined by the end of the Bowman's layer, at the level of vascularized connective tissue. Peripheral cornea was defined 2 mm from limbal landmark, characterized by the avascular cornea. Each of these three portions was cut into cubes of approximately 1 × 1.5 × 2.5 mm by a scalpel, and pooled samples (four squares for specimen) were directly extracted in lysis buffer (mirVana PARIS RNA and Native Protein Purification kit; ThermoFisher Scientific, Waltham, MA, USA). Subsequently, two-thirds of the extract was used for the biochemical analysis and one-third of the extract was devoted to the molecular analysis. Opening quantification analysis was carried out for protein (3-µL extracts) and total RNA (1.5-µL extracts; A280 program; limit as >1.8 ratio) by using a spectrophotometer for small volumes (A1000 Nanodrop; Celbio, Milan, Italy).

Micera A., Jirsova K., Esposito G., Balzamino B.O., Di Zazzo A, & Bonini S. (2020). Mast Cells Populate the Corneoscleral Limbus: New Insights for Our Understanding of Limbal Microenvironment. Investigative Ophthalmology & Visual Science, 61(3), 43.

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Quantifying Protein Expression via Western Blot

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Total protein was extracted using a commercially available mirVana PARIS RNA and Native Protein Purification kit (Thermo Fisher Scientific). Protein levels were detected using appropriate primary antibody dilutions against MOG (1:1000; sourced from Abcam, ab109746) and secondary antibody (Alexa 594-conjugated antibody to rabbit, 1:10 000, Life Technologies, Thermo Fisher Scientific). Primary antibody against alpha tubulin (sourced from Sigma-Aldrich, Cat# T5168) was used to determine equal protein loading. Quantification of protein bands was analysed using Image Studio Lite (LI-COR, Lincoln, NE, USA). Significance was determined by a one-way analysis of variance.

Hoban A.E., Stilling R.M., Ryan F.J., Shanahan F., Dinan T.G., Claesson M.J., Clarke G, & Cryan J.F. (2016). Regulation of prefrontal cortex myelination by the microbiota. Translational Psychiatry, 6(4), e774-.

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Serum miRNA Isolation and Quantification

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Blood samples were obtained by venipuncture using Serum Gel S/7.5 ml collection tubes (Sarstedt S-Monovette), allowed to clot for 60 min at room temperature, and then centrifuged at 1300 ×g for 10 min at 4°C. Next, 500 µl of serum was aliquoted into 1.5 ml siliconized polypropylene microtubes (Sigma-Aldrich, T4816) and stored at -80°C until further use. MiRNAs were isolated from 1200 μl of serum mirVana Paris RNA and Native Protein Purification Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The quantity of miRNA was checked using the MyQubit microRNA Assay (Thermo Fisher Scientific). The small RNA fraction was detected using the Small RNA Kit (Agilent) and a Bioanalyzer 2100. Samples that passed the quality check were kept at -80°C until further analyses.

Kosela-Paterczyk H., Paziewska A., Kulecka M., Balabas A., Kluska A., Dabrowska M., Piatkowska M., Zeber-Lubecka N., Ambrozkiewicz F., Karczmarski J., Mikula M., Rutkowski P, & Ostrowski J. (2020). Signatures of circulating microRNA in four sarcoma subtypes. Journal of Cancer, 11(4), 874-882.

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CSF RNA Isolation and miRNA Detection

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Total RNA (not enriched for small RNAs) was isolated from 0.5 mL CSF using the mirVana™ PARIS™ RNA and Native Protein Purification Kit (ThermoFisher Scientific), with modifications [13 (link)]. RNA samples were concentrated (RNA Clean & Concentrator™−5 Kit; Zymo Research) and eluted into 8 µL of RNAse/DNase-free water. 3.2 µL of concentrated RNA was transcribed using MultiScribe^™Reverse Transcriptase (ThermoFisher Scientific) in an 8 µL reaction volume, and 2.5 µL of the cDNA was pre-amplified in separate reactions with Megaplex™ RT Primer Pool Set v3.0.A or v3.0.B on a T-100 thermocycler (Bio-Rad, Hercules, CA) following the manufacturer’s instructions for detection of miRNAs with pre-amplification. The pre-amplification products were diluted into a prescribed final volume of 100 µL with water, and stored at −20°C.

Lusardi T.A., Wiedrick J.T., Malone M., Phillips J.I., Sandau U.S., Lind B., Quinn J.F., Lapidus J.A, & Saugstad J.A. (2018). Analytics of Cerebrospinal Fluid MicroRNA Quantitative PCR Studies. Molecular neurobiology, 56(7), 4988-4999.

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miRNA Expression Profiling Protocol

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Sample’s total RNA was extracted using mirVana™ PARIS™ RNA and Native Protein Purification Kit (ThermoFisher, MA, USA) according to the directions. TaqMan™ Advanced miRNA cDNA Synthesis Kit was used to perform reverse transcriptase reactions, augment, and qPCR (ThermoFisher, MA, USA). First, the samples were performed the poly(A) tailing reaction, and then performed the adaptor ligation reaction. Next, the miR-Amp reaction was performed followed by the reverse transcription (RT) reaction carried out. Finally, real-time PCR was performed under the experiment settings and PCR thermal cycling conditions (Enzyme activation was at 95°C for 20 s, 1 cycle; Denature was at 95°C for 1 s and Anneal/Extend was at 60°C for 20 s, 40 cycles). The ΔΔCT method was used for quantitative analysis. The quantitative measures were normalized to U6 mRNA levels.

Tang J., Jin L., Liu Y., Li L., Ma Y., Lu L., Ma J., Ding P., Yang X., Liu J, & Yang J. (2020). Exosomes Derived from Mesenchymal Stem Cells Protect the Myocardium Against Ischemia/Reperfusion Injury Through Inhibiting Pyroptosis. Drug Design, Development and Therapy, 14, 3765-3775.

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Total RNA Extraction and Purification

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Total RNA was extracted from blood samples using PureLink™ RNA extraction kit (cat. no. 12183020; Thermo Fisher Scientific, Inc.) and purified using mirVana™ PARIS™ RNA and Native Protein Purification kit (cat. no. AM1556, Ambion; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA integration was determined using an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.). A sample was considered qualified with an RNA integrity number >7.

Hu F., Liu H., Wang C., Li H, & Qiao L. (2021). Expression of the microRNA-30 family in pulmonary arterial hypertension and the role of microRNA-30d-5p in the regulation of pulmonary arterial smooth muscle cell toxicity and apoptosis. Experimental and Therapeutic Medicine, 23(1), 108.

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Quantifying Autophagy Markers in HEK293T

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HEK293T cells, seeded at 600,000 cells/well in 6 well plates, were transfected after 24 hours from seeding using SQSTM1 and MAP1LC3B shRNA plasmid DNA (from Sigma-Aldrich) and Lipofectamine® 2000 transfection reagent (Life Technologies 11668019) according to manufacturer's protocol. Cells were harvested 48 hours after transfection. RNA was purified using the MirVana PARIS RNA and native protein purification kit (Thermo Fisher AM1556), following the manufacturer’s instruction, which allowed purification of RNA and proteins from the same sample for real-time quantitative PCR, TR-FRET and western blot analyses. RNA was further purified from DNA traces by performing a treatment with TURBO DNA-free kit (Ambion, AM1907). Messenger RNA was retrotranscripted using the superscript III first-strand synthesis system for RT-PCR (Life Technologies 18080–051) according to manufacturer's protocol. The power SYBR green PCR master mix (ThermoFisher, 4367659) was applied to test p62 and LC3B expression with the T900 HT AbiPrism real-time PCR Instrument. Expression of p62 and LC3B were calculated and compared with the expression of β-actin through the 2^-ΔΔCT approach and expressed relative to the scramble group.

Bresciani A., Spiezia M.C., Boggio R., Cariulo C., Nordheim A., Altobelli R., Kuhlbrodt K., Dominguez C., Munoz-Sanjuan I., Wityak J., Fodale V., Marchionini D.M, & Weiss A. (2018). Quantifying autophagy using novel LC3B and p62 TR-FRET assays. PLoS ONE, 13(3), e0194423.

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Whey miRNA Isolation using mirVana Kit

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Isolation of miRNA from 300 μl whey/aliquot was performed using the mirVana™ PARIS™ RNA and Native Protein Purification Kit (Thermo Fischer Scientific, Invitrogen, Vinius, Lithuania) and the instructions provided by the manufacturer. The concentration and purity of the miRNA solution were determined by measuring its absorbance at 260 and 280 nm (Additional files 1 and 2).

Jadhav A.B., Ingole S.D., Bharucha S.V., Yoshitha K.L., Gaikwad R.V., Pharande R.R, & Kharde S.D. (2024). Milk miRNA expression in buffaloes as a potential biomarker for mastitis. BMC Veterinary Research, 20, 150.

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CSF miRNA Extraction and Quantification

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Total RNA was extracted from 0.5 mL of each CSF sample using the mirVana™ PARIS™ RNA and Native Protein Purification Kit (ThermoFisher Scientific), modified to include two aqueous extractions during the organic phase extraction steps in order to maximize RNA recovery [13 (link)]. RNA samples were concentrated using the RNA Clean & Concentrator™-5 Kit (Zymo Research) and eluted in 9 µL RNAse/DNase-free water. RNA concentrations were initially measured on a set of test CSF samples using the Quant-iT™ RiboGreen® RNA Assay Kit (ThermoFisher Scientific). The average concentration for the test group was 133 pg/µL with a total RNA recovery of approximately 2 ng/mL CSF. The concentrated RNA samples were converted to cDNA and pre-amplified using a T-100 thermocycler (Bio-Rad, Hercules, CA) with Megaplex™ RT Primers, Human Pool Set v3.0, as per the manufacturer’s protocol (“Megaplex Pools For microRNA Expression Analysis”), following instructions for detection of miRNA with pre-amplification. The pre-amplification products were diluted into a prescribed final volume of 100 µL and stored at −20°C until ready for the final detection PCR reactions. Real-time PCR reactions followed the manufacturer’s protocol, using 18 µL of diluted pre-amplification product.

Lusardi T.A., Phillips J.I., Wiedrick J.T., Harrington C.A., Lind B., Lapidus J.A., Quinn J.F, & Saugstad J.A. (2017). MicroRNAs in Human Cerebrospinal Fluid as Biomarkers for Alzheimer’s Disease. Journal of Alzheimer's disease : JAD, 55(3), 1223-1233.

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