proteinase k solution  (Thermo Fisher)


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    Proteinase K Solution 20 mg mL
    Description:
    Proteinase K from the fungus Engyodontium album is a nonspecific serine protease that is useful for general digestion of proteins Proteinase K remains active • Over a wide pH range optimal activity between 6 5 and 9 5• Under denaturing conditions e g in the presence of SDS or urea• In the presence of metal chelating agents e g EDTA• At comparatively high temperatures optimum digestion temperature is 65°CApplicationsRemoval of endogenous nucleases during the preparation of DNA and RNA preparation of tissue sections for in situ hybridization Performance and quality testingEndodeoxyribonuclease and exodeoxyribonuclease assays liquid form tested for absence of RNase activity Unit definitionOne mAnson unit is described as that amount of enzyme that liberates 1 µmole of Folin positive amino acid within 1 min at 37°C using hemoglobin as a substrate
    Catalog Number:
    25530049
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    None
    Category:
    Proteins Enzymes Peptides
    Applications:
    ChIP-on-Chip|Chromatin Immunoprecipitation (ChIP)|DNA & RNA Purification & Analysis|DNA Extraction|General RNA Purification Reagents & Accessories|General gDNA Purification Reagents & Accessories|Genomic DNA Purification|RNAi, Epigenetics & Non-Coding RNA Research|RNA Extraction|Total RNA Isolation|Chromatin Biology
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    Structured Review

    Thermo Fisher proteinase k solution
    <t>Proteinase</t> K and RNase A sensitivity of the microRNA-enriched 12,000 g and 35,000 g EVs and associated microRNAs. (a–b) Fractions F7 and F8 from the 12,000 g and 35,000 g IDGs were subjected to proteinase K digestion and analysed by transmission electron microscopy (TEM). (c–d) A pool of IDG fractions 7 and 8 from both 12,000 g and 35,000 g IDGs were submitted to proteinase K and/or RNase A digestion, and microRNA bta-miR-223 and bta-miR-125b levels were determined by RT-qPCR. MicroRNA levels are expressed as fold change versus the untreated control (mean ± SD, n = 3) and analysed by comparison with the untreated control (–) by two-tailed paired t -test. (e–f). The leftover proteins from the previous RNA isolation procedure were precipitated following the manufacturer’s protocol and loaded in a 10% acrylamide gel for Western blot detection of TSG-101, XDH and CD63 EV-associated proteins. The signal intensity of each band was measured using ImageJ software. Each band intensity was reported on its non-digested counterpart and is expressed as a percentage of non-treated control (mean ± SD; n = 3). The most representative of the three replicate is displayed above each quantification diagram. The statistical significance of the differences observed was assessed by comparison with the untreated control (–) by a two-tailed paired t -test, * p
    Proteinase K from the fungus Engyodontium album is a nonspecific serine protease that is useful for general digestion of proteins Proteinase K remains active • Over a wide pH range optimal activity between 6 5 and 9 5• Under denaturing conditions e g in the presence of SDS or urea• In the presence of metal chelating agents e g EDTA• At comparatively high temperatures optimum digestion temperature is 65°CApplicationsRemoval of endogenous nucleases during the preparation of DNA and RNA preparation of tissue sections for in situ hybridization Performance and quality testingEndodeoxyribonuclease and exodeoxyribonuclease assays liquid form tested for absence of RNase activity Unit definitionOne mAnson unit is described as that amount of enzyme that liberates 1 µmole of Folin positive amino acid within 1 min at 37°C using hemoglobin as a substrate
    https://www.bioz.com/result/proteinase k solution/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    proteinase k solution - by Bioz Stars, 2021-03
    99/100 stars

    Images

    1) Product Images from "A subset of extracellular vesicles carries the bulk of microRNAs in commercial dairy cow’s milk"

    Article Title: A subset of extracellular vesicles carries the bulk of microRNAs in commercial dairy cow’s milk

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2017.1401897

    Proteinase K and RNase A sensitivity of the microRNA-enriched 12,000 g and 35,000 g EVs and associated microRNAs. (a–b) Fractions F7 and F8 from the 12,000 g and 35,000 g IDGs were subjected to proteinase K digestion and analysed by transmission electron microscopy (TEM). (c–d) A pool of IDG fractions 7 and 8 from both 12,000 g and 35,000 g IDGs were submitted to proteinase K and/or RNase A digestion, and microRNA bta-miR-223 and bta-miR-125b levels were determined by RT-qPCR. MicroRNA levels are expressed as fold change versus the untreated control (mean ± SD, n = 3) and analysed by comparison with the untreated control (–) by two-tailed paired t -test. (e–f). The leftover proteins from the previous RNA isolation procedure were precipitated following the manufacturer’s protocol and loaded in a 10% acrylamide gel for Western blot detection of TSG-101, XDH and CD63 EV-associated proteins. The signal intensity of each band was measured using ImageJ software. Each band intensity was reported on its non-digested counterpart and is expressed as a percentage of non-treated control (mean ± SD; n = 3). The most representative of the three replicate is displayed above each quantification diagram. The statistical significance of the differences observed was assessed by comparison with the untreated control (–) by a two-tailed paired t -test, * p
    Figure Legend Snippet: Proteinase K and RNase A sensitivity of the microRNA-enriched 12,000 g and 35,000 g EVs and associated microRNAs. (a–b) Fractions F7 and F8 from the 12,000 g and 35,000 g IDGs were subjected to proteinase K digestion and analysed by transmission electron microscopy (TEM). (c–d) A pool of IDG fractions 7 and 8 from both 12,000 g and 35,000 g IDGs were submitted to proteinase K and/or RNase A digestion, and microRNA bta-miR-223 and bta-miR-125b levels were determined by RT-qPCR. MicroRNA levels are expressed as fold change versus the untreated control (mean ± SD, n = 3) and analysed by comparison with the untreated control (–) by two-tailed paired t -test. (e–f). The leftover proteins from the previous RNA isolation procedure were precipitated following the manufacturer’s protocol and loaded in a 10% acrylamide gel for Western blot detection of TSG-101, XDH and CD63 EV-associated proteins. The signal intensity of each band was measured using ImageJ software. Each band intensity was reported on its non-digested counterpart and is expressed as a percentage of non-treated control (mean ± SD; n = 3). The most representative of the three replicate is displayed above each quantification diagram. The statistical significance of the differences observed was assessed by comparison with the untreated control (–) by a two-tailed paired t -test, * p

    Techniques Used: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Quantitative RT-PCR, Two Tailed Test, Isolation, Acrylamide Gel Assay, Western Blot, Software

    2) Product Images from "Optimisation of laboratory methods for whole transcriptomic RNA analyses in human left ventricular biopsies and blood samples of clinical relevance"

    Article Title: Optimisation of laboratory methods for whole transcriptomic RNA analyses in human left ventricular biopsies and blood samples of clinical relevance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0213685

    Spectrophotometry analysis of pig LV biopsy RNA. RNA extracted according to each manufacturer’s protocol, with the addition of a proteinase K digestion in half the samples. n = 3 biopsies/experimental condition, mean±SEM. N . B . Exiqon miRCURY tissue protocol requires proteinase K, therefore no–proteinase K condition was done with this kit. (A) RNA yield, assessed as ng RNA recovered per mg input tissue, was not significantly different between the protocols (Kruskal-Wallis test with Dunn correction), and additional proteinase K digestion in the RNAqueous micro, mir Vana and miRNeasy micro protocols did not significantly improve RNA yield (Kruskal-Wallis test with Dunn correction). (B) 260/280 and 260/230 ratios provide an assessment of the purity of the RNA. Ratio
    Figure Legend Snippet: Spectrophotometry analysis of pig LV biopsy RNA. RNA extracted according to each manufacturer’s protocol, with the addition of a proteinase K digestion in half the samples. n = 3 biopsies/experimental condition, mean±SEM. N . B . Exiqon miRCURY tissue protocol requires proteinase K, therefore no–proteinase K condition was done with this kit. (A) RNA yield, assessed as ng RNA recovered per mg input tissue, was not significantly different between the protocols (Kruskal-Wallis test with Dunn correction), and additional proteinase K digestion in the RNAqueous micro, mir Vana and miRNeasy micro protocols did not significantly improve RNA yield (Kruskal-Wallis test with Dunn correction). (B) 260/280 and 260/230 ratios provide an assessment of the purity of the RNA. Ratio

    Techniques Used: Spectrophotometry

    Summary of experimental design for Figs 2 , 3 and 4 . (A) Left ventricular biopsies were taken from three pigs undergoing cardio-pulmonary bypass (CPB). 7 biopsies were taken per pig. RNA was isolated from the biopsies using four commercially available kits; RNAqueous micro, mir Vana, miRCURY tissue, miRNeasy micro. Three kits (RNAqueous micro, mir Vana, miRNeasy micro) were tested with and without an additional proteinase K digestion step, giving 7 protocols in total. The miRCURY tissue protocol includes a proteinase K digestion, therefore this kit was not tested without this. RNA samples were characterised by spectrophotometry and capillary electrophoresis. (B) Blood samples were taken from three pigs undergoing CPB and processed to plasma. RNA was isolated from the plasma using four commercially-available kits; miRNeasy serum/plasma, mir Vana, miRCURY biofluids, Norgen Biotek plasma/serum. Each kit was tested with and without glycogen as a co-precipitant, giving 8 protocols in total. RNA samples were evaluated by RT-qPCR for the exogenous spike-in cel-miR-39-3p, and endogenous miRNAs miR-16-5p, miR-21-5p, miR-92a-3p.
    Figure Legend Snippet: Summary of experimental design for Figs 2 , 3 and 4 . (A) Left ventricular biopsies were taken from three pigs undergoing cardio-pulmonary bypass (CPB). 7 biopsies were taken per pig. RNA was isolated from the biopsies using four commercially available kits; RNAqueous micro, mir Vana, miRCURY tissue, miRNeasy micro. Three kits (RNAqueous micro, mir Vana, miRNeasy micro) were tested with and without an additional proteinase K digestion step, giving 7 protocols in total. The miRCURY tissue protocol includes a proteinase K digestion, therefore this kit was not tested without this. RNA samples were characterised by spectrophotometry and capillary electrophoresis. (B) Blood samples were taken from three pigs undergoing CPB and processed to plasma. RNA was isolated from the plasma using four commercially-available kits; miRNeasy serum/plasma, mir Vana, miRCURY biofluids, Norgen Biotek plasma/serum. Each kit was tested with and without glycogen as a co-precipitant, giving 8 protocols in total. RNA samples were evaluated by RT-qPCR for the exogenous spike-in cel-miR-39-3p, and endogenous miRNAs miR-16-5p, miR-21-5p, miR-92a-3p.

    Techniques Used: Isolation, Spectrophotometry, Electrophoresis, Quantitative RT-PCR

    3) Product Images from "Optimization of PCR for quantification of simian immunodeficiency virus (SIV) genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA"

    Article Title: Optimization of PCR for quantification of simian immunodeficiency virus (SIV) genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA

    Journal: Journal of medical primatology

    doi: 10.1111/jmp.12088

    Effects of modifications applied to vRNA extraction and isolation. Panel A . Addition of Proteinase K at 1.0 and 2.5 mg / ml to SIVmac239-containing plasma followed by extraction using the Roche High Pure Viral Kit, cDNA synthesis (using 15 μl
    Figure Legend Snippet: Effects of modifications applied to vRNA extraction and isolation. Panel A . Addition of Proteinase K at 1.0 and 2.5 mg / ml to SIVmac239-containing plasma followed by extraction using the Roche High Pure Viral Kit, cDNA synthesis (using 15 μl

    Techniques Used: Isolation

    Related Articles

    Fluorescence In Situ Hybridization:

    Article Title: An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure
    Article Snippet: Accell SMART Pool siRNAs and ON-TARGETplus SMART Pool siRNAs listed in table S were purchased from GE Healthcare Dharmacon. .. All other reagents were obtained from the following sources: Bafilomycin A1 (LC Laboratories, B-1080); bovine serum albumin (BSA) (EMD Millipore, 126575; Calbiochem, 126575 (for IEM)); digitonin (Sigma-Aldrich, D141); gelatin from cold water fish skin (Sigma-Aldrich, G7765); GoldEnhance EM (Nanoprobes, 2113); Membrane-impermeable HaloTag Ligand (MIL) (Promega, Alexa Fluor 488-conjugated, G1001; Alexa Fluor 660-conjugated, G8471); Membrane-permeable HaloTag Ligand (MPL) (Promega, tetramethylrhodamine-conjugated, G8251); normal goat serum (Sigma-Aldrich, G9023); Nucleofector Kit V (Lonza, VCA-1003); Nucleofector Kit R (Lonza, VCA-1001); paraformaldehyde (Electron Microscopy Sciences, 15710); proteinase K (Invitrogen, 25530-049); saponin (Sigma-Aldrich, 84510); thapsigargin (Sigma-Aldrich, T9033); XF Plasma Membrane Permeabilizer (XF-PMP) (Seahorse Bioscience, 102504-100). pHaloTag-human MAP1LC3-Lv110 (HT-LC3) was custom-made by GeneCopoeia. .. The human CHMP2A cDNA (gift from Dr. Gerlich, Addgene#31805) was amplified by PCR using a primer set (5′–TTTGCTAGCGCCACCATGGACCTATTGTTCGGGC-3′; 5′-TTGAATTCGGTCCCTCCGCAGGTTCTTAA-3′) and subcloned into the Nhe I-EcoRI site of pCDH1-EGFP(N1)-EF1-puro.

    Electron Microscopy:

    Article Title: An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure
    Article Snippet: Accell SMART Pool siRNAs and ON-TARGETplus SMART Pool siRNAs listed in table S were purchased from GE Healthcare Dharmacon. .. All other reagents were obtained from the following sources: Bafilomycin A1 (LC Laboratories, B-1080); bovine serum albumin (BSA) (EMD Millipore, 126575; Calbiochem, 126575 (for IEM)); digitonin (Sigma-Aldrich, D141); gelatin from cold water fish skin (Sigma-Aldrich, G7765); GoldEnhance EM (Nanoprobes, 2113); Membrane-impermeable HaloTag Ligand (MIL) (Promega, Alexa Fluor 488-conjugated, G1001; Alexa Fluor 660-conjugated, G8471); Membrane-permeable HaloTag Ligand (MPL) (Promega, tetramethylrhodamine-conjugated, G8251); normal goat serum (Sigma-Aldrich, G9023); Nucleofector Kit V (Lonza, VCA-1003); Nucleofector Kit R (Lonza, VCA-1001); paraformaldehyde (Electron Microscopy Sciences, 15710); proteinase K (Invitrogen, 25530-049); saponin (Sigma-Aldrich, 84510); thapsigargin (Sigma-Aldrich, T9033); XF Plasma Membrane Permeabilizer (XF-PMP) (Seahorse Bioscience, 102504-100). pHaloTag-human MAP1LC3-Lv110 (HT-LC3) was custom-made by GeneCopoeia. .. The human CHMP2A cDNA (gift from Dr. Gerlich, Addgene#31805) was amplified by PCR using a primer set (5′–TTTGCTAGCGCCACCATGGACCTATTGTTCGGGC-3′; 5′-TTGAATTCGGTCCCTCCGCAGGTTCTTAA-3′) and subcloned into the Nhe I-EcoRI site of pCDH1-EGFP(N1)-EF1-puro.

    Incubation:

    Article Title: Multiple Megaplasmids Confer Extremely High Levels of Metal Tolerance in Alteromonas Strains
    Article Snippet: .. The plugs were then incubated in 4 ml of proteinase K solution (100 mM EDTA [pH 8], 0.2% Na deoxycholate, 1% Na lauryl sarcosine, with 1 mg/ml proteinase K [Fisher]) at 50°C overnight. ..

    Article Title: A subset of extracellular vesicles carries the bulk of microRNAs in commercial dairy cow’s milk
    Article Snippet: .. Briefly, milk EVs were incubated with proteinase K (0.05 µg/µL, AM2546; Ambion, Foster City, CA, USA) for 10 min at 37°C. .. Phenylmethylsulfonyl fluoride (PMSF, 5 mM; Sigma-Aldrich) was then added and the incubation followed for an additional 10 min at room temperature; further proteinase K inhibition was ensured by heating the samples at 90°C for 5 min.

    Article Title: Optimization of PCR for quantification of simian immunodeficiency virus (SIV) genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA
    Article Snippet: The centrifugation step allows for quantification of virus from higher volumes of plasma when PVL is expected to be low. .. To inactivate nucleases and other contaminants, proteinase K (cat. #AM2548, Life Technologies; Carlsbad, CA) was added to neat plasma or virus pellet specimens at a final concentration of 2.5 mg/ml followed by incubation at 37°C for 30 min to inactivate further proteolysis [ ]. .. Viral RNA (vRNA) was extracted from plasma using High Pure Viral RNA kit (cat. #11858882001, Roche; Indianapolis, IN) using the manufacture’s protocol with the modification that the elution volume was increased from 50 μl to 100 μl and supplemented with RNase Inhibitor (0.3 units/μl; cat. #N8080119, Life Technologies).

    Article Title: The RNA-binding protein hnRNPU regulates the sorting of microRNA-30c-5p into large extracellular vesicles
    Article Snippet: Vesicular hnRNPU degradation assay In order to confirm the presence of hnRNPU inside of EVs, EVs were isolated from 2.4 × 107 HCAECs per sample, as described above and then resuspended in 10 µL PBS or 1% Triton X-100. .. After 10 s of vortexing and incubation at RT for 5 min, half of the PBS-treated samples as well as the Triton X-treated samples were incubated with 5 µL proteinase K (20 mg/mL) solution (Thermo Fisher Scientific, Cat. # 25-530-049). ..

    Concentration Assay:

    Article Title: Optimization of PCR for quantification of simian immunodeficiency virus (SIV) genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA
    Article Snippet: The centrifugation step allows for quantification of virus from higher volumes of plasma when PVL is expected to be low. .. To inactivate nucleases and other contaminants, proteinase K (cat. #AM2548, Life Technologies; Carlsbad, CA) was added to neat plasma or virus pellet specimens at a final concentration of 2.5 mg/ml followed by incubation at 37°C for 30 min to inactivate further proteolysis [ ]. .. Viral RNA (vRNA) was extracted from plasma using High Pure Viral RNA kit (cat. #11858882001, Roche; Indianapolis, IN) using the manufacture’s protocol with the modification that the elution volume was increased from 50 μl to 100 μl and supplemented with RNase Inhibitor (0.3 units/μl; cat. #N8080119, Life Technologies).

    Lysis:

    Article Title: Pharmacokinetic Profiling of Conjugated Therapeutic Oligonucleotides: A High-Throughput Method Based Upon Serial Blood Microsampling Coupled to Peptide Nucleic Acid Hybridization Assay
    Article Snippet: .. Briefly, blood and plasma samples were further diluted in lysis solution to a total of 200 μL (Cat. No. MTC096H; MasterPure, EpiCentre) containing 1 μL proteinase K (20 mg/mL) (Cat. No. 25530-049; Invitrogen). .. Alternatively, liver, kidney, and spleen samples were lysed and digested in 300 μL of QG Homogenizing Solution (Cat. No. QG0517; Affymetrix) containing 1–2 μL proteinase K (20 mg/mL).

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    Thermo Fisher proteinase k solution
    <t>Proteinase</t> K and RNase A sensitivity of the microRNA-enriched 12,000 g and 35,000 g EVs and associated microRNAs. (a–b) Fractions F7 and F8 from the 12,000 g and 35,000 g IDGs were subjected to proteinase K digestion and analysed by transmission electron microscopy (TEM). (c–d) A pool of IDG fractions 7 and 8 from both 12,000 g and 35,000 g IDGs were submitted to proteinase K and/or RNase A digestion, and microRNA bta-miR-223 and bta-miR-125b levels were determined by RT-qPCR. MicroRNA levels are expressed as fold change versus the untreated control (mean ± SD, n = 3) and analysed by comparison with the untreated control (–) by two-tailed paired t -test. (e–f). The leftover proteins from the previous RNA isolation procedure were precipitated following the manufacturer’s protocol and loaded in a 10% acrylamide gel for Western blot detection of TSG-101, XDH and CD63 EV-associated proteins. The signal intensity of each band was measured using ImageJ software. Each band intensity was reported on its non-digested counterpart and is expressed as a percentage of non-treated control (mean ± SD; n = 3). The most representative of the three replicate is displayed above each quantification diagram. The statistical significance of the differences observed was assessed by comparison with the untreated control (–) by a two-tailed paired t -test, * p
    Proteinase K Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteinase k solution/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    proteinase k solution - by Bioz Stars, 2021-03
    99/100 stars
      Buy from Supplier

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    Proteinase K and RNase A sensitivity of the microRNA-enriched 12,000 g and 35,000 g EVs and associated microRNAs. (a–b) Fractions F7 and F8 from the 12,000 g and 35,000 g IDGs were subjected to proteinase K digestion and analysed by transmission electron microscopy (TEM). (c–d) A pool of IDG fractions 7 and 8 from both 12,000 g and 35,000 g IDGs were submitted to proteinase K and/or RNase A digestion, and microRNA bta-miR-223 and bta-miR-125b levels were determined by RT-qPCR. MicroRNA levels are expressed as fold change versus the untreated control (mean ± SD, n = 3) and analysed by comparison with the untreated control (–) by two-tailed paired t -test. (e–f). The leftover proteins from the previous RNA isolation procedure were precipitated following the manufacturer’s protocol and loaded in a 10% acrylamide gel for Western blot detection of TSG-101, XDH and CD63 EV-associated proteins. The signal intensity of each band was measured using ImageJ software. Each band intensity was reported on its non-digested counterpart and is expressed as a percentage of non-treated control (mean ± SD; n = 3). The most representative of the three replicate is displayed above each quantification diagram. The statistical significance of the differences observed was assessed by comparison with the untreated control (–) by a two-tailed paired t -test, * p

    Journal: Journal of Extracellular Vesicles

    Article Title: A subset of extracellular vesicles carries the bulk of microRNAs in commercial dairy cow’s milk

    doi: 10.1080/20013078.2017.1401897

    Figure Lengend Snippet: Proteinase K and RNase A sensitivity of the microRNA-enriched 12,000 g and 35,000 g EVs and associated microRNAs. (a–b) Fractions F7 and F8 from the 12,000 g and 35,000 g IDGs were subjected to proteinase K digestion and analysed by transmission electron microscopy (TEM). (c–d) A pool of IDG fractions 7 and 8 from both 12,000 g and 35,000 g IDGs were submitted to proteinase K and/or RNase A digestion, and microRNA bta-miR-223 and bta-miR-125b levels were determined by RT-qPCR. MicroRNA levels are expressed as fold change versus the untreated control (mean ± SD, n = 3) and analysed by comparison with the untreated control (–) by two-tailed paired t -test. (e–f). The leftover proteins from the previous RNA isolation procedure were precipitated following the manufacturer’s protocol and loaded in a 10% acrylamide gel for Western blot detection of TSG-101, XDH and CD63 EV-associated proteins. The signal intensity of each band was measured using ImageJ software. Each band intensity was reported on its non-digested counterpart and is expressed as a percentage of non-treated control (mean ± SD; n = 3). The most representative of the three replicate is displayed above each quantification diagram. The statistical significance of the differences observed was assessed by comparison with the untreated control (–) by a two-tailed paired t -test, * p

    Article Snippet: Briefly, milk EVs were incubated with proteinase K (0.05 µg/µL, AM2546; Ambion, Foster City, CA, USA) for 10 min at 37°C.

    Techniques: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Quantitative RT-PCR, Two Tailed Test, Isolation, Acrylamide Gel Assay, Western Blot, Software

    Spectrophotometry analysis of pig LV biopsy RNA. RNA extracted according to each manufacturer’s protocol, with the addition of a proteinase K digestion in half the samples. n = 3 biopsies/experimental condition, mean±SEM. N . B . Exiqon miRCURY tissue protocol requires proteinase K, therefore no–proteinase K condition was done with this kit. (A) RNA yield, assessed as ng RNA recovered per mg input tissue, was not significantly different between the protocols (Kruskal-Wallis test with Dunn correction), and additional proteinase K digestion in the RNAqueous micro, mir Vana and miRNeasy micro protocols did not significantly improve RNA yield (Kruskal-Wallis test with Dunn correction). (B) 260/280 and 260/230 ratios provide an assessment of the purity of the RNA. Ratio

    Journal: PLoS ONE

    Article Title: Optimisation of laboratory methods for whole transcriptomic RNA analyses in human left ventricular biopsies and blood samples of clinical relevance

    doi: 10.1371/journal.pone.0213685

    Figure Lengend Snippet: Spectrophotometry analysis of pig LV biopsy RNA. RNA extracted according to each manufacturer’s protocol, with the addition of a proteinase K digestion in half the samples. n = 3 biopsies/experimental condition, mean±SEM. N . B . Exiqon miRCURY tissue protocol requires proteinase K, therefore no–proteinase K condition was done with this kit. (A) RNA yield, assessed as ng RNA recovered per mg input tissue, was not significantly different between the protocols (Kruskal-Wallis test with Dunn correction), and additional proteinase K digestion in the RNAqueous micro, mir Vana and miRNeasy micro protocols did not significantly improve RNA yield (Kruskal-Wallis test with Dunn correction). (B) 260/280 and 260/230 ratios provide an assessment of the purity of the RNA. Ratio

    Article Snippet: Thereafter, the manufacturer’s protocol was followed exactly in one set of extractions, while in a parallel set of extractions the protocol was modified to include a proteinase K digestion step; samples were incubated with 200 μg/mL proteinase K (Invitrogen AM2546) for 15 minutes at 55°C.

    Techniques: Spectrophotometry

    Summary of experimental design for Figs 2 , 3 and 4 . (A) Left ventricular biopsies were taken from three pigs undergoing cardio-pulmonary bypass (CPB). 7 biopsies were taken per pig. RNA was isolated from the biopsies using four commercially available kits; RNAqueous micro, mir Vana, miRCURY tissue, miRNeasy micro. Three kits (RNAqueous micro, mir Vana, miRNeasy micro) were tested with and without an additional proteinase K digestion step, giving 7 protocols in total. The miRCURY tissue protocol includes a proteinase K digestion, therefore this kit was not tested without this. RNA samples were characterised by spectrophotometry and capillary electrophoresis. (B) Blood samples were taken from three pigs undergoing CPB and processed to plasma. RNA was isolated from the plasma using four commercially-available kits; miRNeasy serum/plasma, mir Vana, miRCURY biofluids, Norgen Biotek plasma/serum. Each kit was tested with and without glycogen as a co-precipitant, giving 8 protocols in total. RNA samples were evaluated by RT-qPCR for the exogenous spike-in cel-miR-39-3p, and endogenous miRNAs miR-16-5p, miR-21-5p, miR-92a-3p.

    Journal: PLoS ONE

    Article Title: Optimisation of laboratory methods for whole transcriptomic RNA analyses in human left ventricular biopsies and blood samples of clinical relevance

    doi: 10.1371/journal.pone.0213685

    Figure Lengend Snippet: Summary of experimental design for Figs 2 , 3 and 4 . (A) Left ventricular biopsies were taken from three pigs undergoing cardio-pulmonary bypass (CPB). 7 biopsies were taken per pig. RNA was isolated from the biopsies using four commercially available kits; RNAqueous micro, mir Vana, miRCURY tissue, miRNeasy micro. Three kits (RNAqueous micro, mir Vana, miRNeasy micro) were tested with and without an additional proteinase K digestion step, giving 7 protocols in total. The miRCURY tissue protocol includes a proteinase K digestion, therefore this kit was not tested without this. RNA samples were characterised by spectrophotometry and capillary electrophoresis. (B) Blood samples were taken from three pigs undergoing CPB and processed to plasma. RNA was isolated from the plasma using four commercially-available kits; miRNeasy serum/plasma, mir Vana, miRCURY biofluids, Norgen Biotek plasma/serum. Each kit was tested with and without glycogen as a co-precipitant, giving 8 protocols in total. RNA samples were evaluated by RT-qPCR for the exogenous spike-in cel-miR-39-3p, and endogenous miRNAs miR-16-5p, miR-21-5p, miR-92a-3p.

    Article Snippet: Thereafter, the manufacturer’s protocol was followed exactly in one set of extractions, while in a parallel set of extractions the protocol was modified to include a proteinase K digestion step; samples were incubated with 200 μg/mL proteinase K (Invitrogen AM2546) for 15 minutes at 55°C.

    Techniques: Isolation, Spectrophotometry, Electrophoresis, Quantitative RT-PCR

    (A) Schematic diagram of the EV isolation protocol used. (B) Immunoblotting of Annexin V (36 kDa), Flotillin-1 (48 kDa), CD9 (26 kDa), CD63 (25 kDa) and β-Actin (42 kDa) in HCAECs and EVs. (C) Size distribution of EVs analysed by nanoparticle tracking analysis (size ~70–500 nm), dotted lines = SEM. (D) Transmission electron microscopic image (85,000×) of pelleted large EVs (size ~100–500 nm). (E) Proteomic analysis of large EVs via mass spectrometry: > 3000 proteins the largest portion of which are annotated as vesicular proteins. (F) Functional annotation reveals a relevant portion of RNA-binding proteins. (G) Ranking of the top 10 RNA-binding proteins found in EVs, sorted by area. (H) Immunoblot of hnRNPU and β-Actin of EVs from HCAECs with and without prior degradation by proteinase K and 1% Triton X-100 confirms the presence of hnRNPU in EVs.

    Journal: Journal of Extracellular Vesicles

    Article Title: The RNA-binding protein hnRNPU regulates the sorting of microRNA-30c-5p into large extracellular vesicles

    doi: 10.1080/20013078.2020.1786967

    Figure Lengend Snippet: (A) Schematic diagram of the EV isolation protocol used. (B) Immunoblotting of Annexin V (36 kDa), Flotillin-1 (48 kDa), CD9 (26 kDa), CD63 (25 kDa) and β-Actin (42 kDa) in HCAECs and EVs. (C) Size distribution of EVs analysed by nanoparticle tracking analysis (size ~70–500 nm), dotted lines = SEM. (D) Transmission electron microscopic image (85,000×) of pelleted large EVs (size ~100–500 nm). (E) Proteomic analysis of large EVs via mass spectrometry: > 3000 proteins the largest portion of which are annotated as vesicular proteins. (F) Functional annotation reveals a relevant portion of RNA-binding proteins. (G) Ranking of the top 10 RNA-binding proteins found in EVs, sorted by area. (H) Immunoblot of hnRNPU and β-Actin of EVs from HCAECs with and without prior degradation by proteinase K and 1% Triton X-100 confirms the presence of hnRNPU in EVs.

    Article Snippet: After 10 s of vortexing and incubation at RT for 5 min, half of the PBS-treated samples as well as the Triton X-treated samples were incubated with 5 µL proteinase K (20 mg/mL) solution (Thermo Fisher Scientific, Cat. # 25-530-049).

    Techniques: Isolation, Transmission Assay, Mass Spectrometry, Functional Assay, RNA Binding Assay

    Effects of modifications applied to vRNA extraction and isolation. Panel A . Addition of Proteinase K at 1.0 and 2.5 mg / ml to SIVmac239-containing plasma followed by extraction using the Roche High Pure Viral Kit, cDNA synthesis (using 15 μl

    Journal: Journal of medical primatology

    Article Title: Optimization of PCR for quantification of simian immunodeficiency virus (SIV) genomic RNA in plasma of rhesus macaques (Macaca mulatta) using armored RNA

    doi: 10.1111/jmp.12088

    Figure Lengend Snippet: Effects of modifications applied to vRNA extraction and isolation. Panel A . Addition of Proteinase K at 1.0 and 2.5 mg / ml to SIVmac239-containing plasma followed by extraction using the Roche High Pure Viral Kit, cDNA synthesis (using 15 μl

    Article Snippet: Reverse transcription (RT) master mix was prepared containing MultiScribe™ Reverse Transcriptase (1.25 units/μl; cat. #4311235, Life Technologies), RNase Inhibitor (0.4 units/μl; Life Technologies), Buffer II (1× final; Life Technologies), MgCl2 (6.5 nM/μl; cat. #N8080130, Life Technologies), dNTPs (2 nM/μl final; cat. #N8080260, Life Technologies), and BSA as a carrier protein (0.5 mg/ml; cat. #AM2548, Life Technologies) for vRNA cDNA synthesis in a final reaction volume of 50 μl.

    Techniques: Isolation