mirneasy mini kit  (Qiagen)

 
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    Name:
    miRNeasy Mini Kit
    Description:
    For purification of microRNA and total RNA from tissues and cells Kit contents Qiagen miRNeasy Mini Kit 50 preps 50 to 100mg Sample 30L Elution Volume Tissue Cells Sample miRNA Total RNA Purification Spin Column Format Silica Technology Ideal for PCR qPCR Real time RT PCR Microarray For Purification of microRNA and Total RNA from Tissues and Cells Includes 50 RNeasy Mini Spin Columns Collection Tubes 1 5 and 2mL QIAzol Lysis Reagent RNase free Reagents and Buffers Benefits Effective purification of miRNA and total RNA Efficient enrichment of miRNA and RNAs 200 nucleotides High purity RNA suitable for all downstream applications Protocols for copurification or isolation of separate fractio
    Catalog Number:
    217004
    Price:
    368
    Category:
    miRNeasy Mini Kit
    Buy from Supplier


    Structured Review

    Qiagen mirneasy mini kit
    miRNeasy Mini Kit
    For purification of microRNA and total RNA from tissues and cells Kit contents Qiagen miRNeasy Mini Kit 50 preps 50 to 100mg Sample 30L Elution Volume Tissue Cells Sample miRNA Total RNA Purification Spin Column Format Silica Technology Ideal for PCR qPCR Real time RT PCR Microarray For Purification of microRNA and Total RNA from Tissues and Cells Includes 50 RNeasy Mini Spin Columns Collection Tubes 1 5 and 2mL QIAzol Lysis Reagent RNase free Reagents and Buffers Benefits Effective purification of miRNA and total RNA Efficient enrichment of miRNA and RNAs 200 nucleotides High purity RNA suitable for all downstream applications Protocols for copurification or isolation of separate fractio
    https://www.bioz.com/result/mirneasy mini kit/product/Qiagen
    Average 99 stars, based on 3913 article reviews
    Price from $9.99 to $1999.99
    mirneasy mini kit - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Controlled ovarian hyperstimulation induced changes in the expression of circulatory miRNA in bovine follicular fluid and blood plasma"

    Article Title: Controlled ovarian hyperstimulation induced changes in the expression of circulatory miRNA in bovine follicular fluid and blood plasma

    Journal: Journal of Ovarian Research

    doi: 10.1186/s13048-015-0208-5

    Specificity of isolation of exosomes and Ago2 protein complex from follicular fluid and blood plasma. Exosome and Ago2 proteins were isolated from organic-phenol fraction during total RNA isolation using miRNeasy kit and resolved in 8M urea. Protein concentrations were quantified using Bradford assay and total of twenty microgram protein from each group were separated in 12 % SDS-PAGE, transferred nitrocellulose membrane and incubated with specific antibody (CD63 and Ago2). Followed by HRP-conjugated secondary antibody and detected using chemiluminescent substrate. Western-blot results indicate the specificity of exosome and Ago2 protein isolation from follicular fluid (FF) and blood plasma (BP) as indicated in the corresponding figures
    Figure Legend Snippet: Specificity of isolation of exosomes and Ago2 protein complex from follicular fluid and blood plasma. Exosome and Ago2 proteins were isolated from organic-phenol fraction during total RNA isolation using miRNeasy kit and resolved in 8M urea. Protein concentrations were quantified using Bradford assay and total of twenty microgram protein from each group were separated in 12 % SDS-PAGE, transferred nitrocellulose membrane and incubated with specific antibody (CD63 and Ago2). Followed by HRP-conjugated secondary antibody and detected using chemiluminescent substrate. Western-blot results indicate the specificity of exosome and Ago2 protein isolation from follicular fluid (FF) and blood plasma (BP) as indicated in the corresponding figures

    Techniques Used: Isolation, Bradford Assay, SDS Page, Incubation, Western Blot

    2) Product Images from "Bias in recent miRBase annotations potentially associated with RNA quality issues"

    Article Title: Bias in recent miRBase annotations potentially associated with RNA quality issues

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-05070-0

    Experimental design. ( a ) Liver, heart and brain of male mice were harvested immediately after death, divided into 8 parts of about equal size, and stored at either 4 °C or at room temperature (RT) for the indicated time periods before RNA isolation. Experiments were performed in biological triplicates. RNA integrity was measured with Bioanalyzer. Gel-like image of brain tissue is given as example. MiRNA expression profiles of one replicate were measured using microarrays. ( b ) Liver tissue of 3 male mice was harvested immediately after death and divided into 5 parts of about equal size. Three parts were immediately transferred into RNAlater (0 h), two parts were stored for 96 h at room temperature (96 h). Two samples (0 h and 96 h) were isolated using standard procedure with miRNeasy Kit without DNase digestion. Two samples (0 h and 96 h) were isolated with optional DNase digestion to exclude DNA background. From the remaining undegraded sample (0 h), total RNA without small RNAs was isolated using RNeasy Kit with optional DNase digestion. Isolated RNA was further treated with 0 U, 0.026 U and 0.67 U RNase for 30 min to generate artificial RNA degradation. RNA integrity was measured with Bioanalyzer. MiRNA expression profiles of all replicates were measured using microarrays. The schematic drawings were prepared using the Biomedical-PPT-Toolkit-Suite from Motifolio Inc., USA.
    Figure Legend Snippet: Experimental design. ( a ) Liver, heart and brain of male mice were harvested immediately after death, divided into 8 parts of about equal size, and stored at either 4 °C or at room temperature (RT) for the indicated time periods before RNA isolation. Experiments were performed in biological triplicates. RNA integrity was measured with Bioanalyzer. Gel-like image of brain tissue is given as example. MiRNA expression profiles of one replicate were measured using microarrays. ( b ) Liver tissue of 3 male mice was harvested immediately after death and divided into 5 parts of about equal size. Three parts were immediately transferred into RNAlater (0 h), two parts were stored for 96 h at room temperature (96 h). Two samples (0 h and 96 h) were isolated using standard procedure with miRNeasy Kit without DNase digestion. Two samples (0 h and 96 h) were isolated with optional DNase digestion to exclude DNA background. From the remaining undegraded sample (0 h), total RNA without small RNAs was isolated using RNeasy Kit with optional DNase digestion. Isolated RNA was further treated with 0 U, 0.026 U and 0.67 U RNase for 30 min to generate artificial RNA degradation. RNA integrity was measured with Bioanalyzer. MiRNA expression profiles of all replicates were measured using microarrays. The schematic drawings were prepared using the Biomedical-PPT-Toolkit-Suite from Motifolio Inc., USA.

    Techniques Used: Mouse Assay, Isolation, Expressing

    3) Product Images from "Optimized microRNA purification from TRIzol-treated plasma"

    Article Title: Optimized microRNA purification from TRIzol-treated plasma

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1299-5

    MiRNA recovery from decreasing amounts of plasma input. The impact of plasma volume on the recovery of (A) exogenous RNA sequences and (B) endogenous miRNAs was evaluated using real-time RT-PCR. Plasma samples were diluted in water to 50 μL and processed with the QIAGEN miRNeasy Mini Kit. In (A) , three synthetic sequences of different amounts were spiked into the samples and measured in six technical replicates. Significance compared to the water-only control was determined using a linear mixed effects model, **** = p
    Figure Legend Snippet: MiRNA recovery from decreasing amounts of plasma input. The impact of plasma volume on the recovery of (A) exogenous RNA sequences and (B) endogenous miRNAs was evaluated using real-time RT-PCR. Plasma samples were diluted in water to 50 μL and processed with the QIAGEN miRNeasy Mini Kit. In (A) , three synthetic sequences of different amounts were spiked into the samples and measured in six technical replicates. Significance compared to the water-only control was determined using a linear mixed effects model, **** = p

    Techniques Used: Quantitative RT-PCR

    MiRNA recovery with 5 μg glycogen or linear acrylamide as a co-precipitant during RNA extraction. A . The effect of glycogen or linear acrylamide (LA) on miRNA recovery was determined for the RNeasy and miRNeasy kits. Boxplot whiskers indicate minimum and maximum values, with the line drawn at the median. Individual datapoints show the spread (n = 6 for each sequence; total n = 18). Extraction efficiencies were compared to the no carrier control sample, and significance was calculated using a linear mixed effects model. **** indicates p
    Figure Legend Snippet: MiRNA recovery with 5 μg glycogen or linear acrylamide as a co-precipitant during RNA extraction. A . The effect of glycogen or linear acrylamide (LA) on miRNA recovery was determined for the RNeasy and miRNeasy kits. Boxplot whiskers indicate minimum and maximum values, with the line drawn at the median. Individual datapoints show the spread (n = 6 for each sequence; total n = 18). Extraction efficiencies were compared to the no carrier control sample, and significance was calculated using a linear mixed effects model. **** indicates p

    Techniques Used: RNA Extraction, Sequencing

    4) Product Images from "Estradiol suppresses NF-?B activation through coordinated regulation of let-7a and miR-125b in primary human macrophages"

    Article Title: Estradiol suppresses NF-?B activation through coordinated regulation of let-7a and miR-125b in primary human macrophages

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0903463

    Let-7a and miR-125b are regulated by LPS (A) Total RNA was extracted from macrophages using Qiagen miRNeasy kits. Let-7a and miR-125b expression were measured using Taqman miRNA assay system. Data are normalized to U6 expression levels. (B) let-7a and (C) miR-125b expression levels in response to LPS stimulation. Data were normalized to U6 and are presented as relative to unstimulated levels. (D–E) Macrophages from a representative donor were treated with NF-κB activation inhibitor (10 μM) for 1 hour prior to LPS stimulation for (D) 12 hours or (E) 3 hours. *p
    Figure Legend Snippet: Let-7a and miR-125b are regulated by LPS (A) Total RNA was extracted from macrophages using Qiagen miRNeasy kits. Let-7a and miR-125b expression were measured using Taqman miRNA assay system. Data are normalized to U6 expression levels. (B) let-7a and (C) miR-125b expression levels in response to LPS stimulation. Data were normalized to U6 and are presented as relative to unstimulated levels. (D–E) Macrophages from a representative donor were treated with NF-κB activation inhibitor (10 μM) for 1 hour prior to LPS stimulation for (D) 12 hours or (E) 3 hours. *p

    Techniques Used: Expressing, TaqMan microRNA Assay, Activation Assay

    5) Product Images from "MicroRNA-205 Directly Regulates the Tumor Suppressor, Interleukin-24, in Human KB Oral Cancer Cells"

    Article Title: MicroRNA-205 Directly Regulates the Tumor Suppressor, Interleukin-24, in Human KB Oral Cancer Cells

    Journal: Molecules and Cells

    doi: 10.1007/s10059-013-2154-7

    MicroRNA array in KB cells compared with NHOK cells. Total RNA from both KB cells and NHOK were isolated with miRNeasy mini kit following the manufacturer’s instructions. The concentration, purity, and amount of total RNA were quantified using
    Figure Legend Snippet: MicroRNA array in KB cells compared with NHOK cells. Total RNA from both KB cells and NHOK were isolated with miRNeasy mini kit following the manufacturer’s instructions. The concentration, purity, and amount of total RNA were quantified using

    Techniques Used: Isolation, Concentration Assay

    6) Product Images from "Optimized microRNA purification from TRIzol-treated plasma"

    Article Title: Optimized microRNA purification from TRIzol-treated plasma

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1299-5

    MiRNA recovery from decreasing amounts of plasma input. The impact of plasma volume on the recovery of (A) exogenous RNA sequences and (B) endogenous miRNAs was evaluated using real-time RT-PCR. Plasma samples were diluted in water to 50 μL and processed with the QIAGEN miRNeasy Mini Kit. In (A) , three synthetic sequences of different amounts were spiked into the samples and measured in six technical replicates. Significance compared to the water-only control was determined using a linear mixed effects model, **** = p
    Figure Legend Snippet: MiRNA recovery from decreasing amounts of plasma input. The impact of plasma volume on the recovery of (A) exogenous RNA sequences and (B) endogenous miRNAs was evaluated using real-time RT-PCR. Plasma samples were diluted in water to 50 μL and processed with the QIAGEN miRNeasy Mini Kit. In (A) , three synthetic sequences of different amounts were spiked into the samples and measured in six technical replicates. Significance compared to the water-only control was determined using a linear mixed effects model, **** = p

    Techniques Used: Quantitative RT-PCR

    MiRNA recovery with 5 μg glycogen or linear acrylamide as a co-precipitant during RNA extraction. A . The effect of glycogen or linear acrylamide (LA) on miRNA recovery was determined for the RNeasy and miRNeasy kits. Boxplot whiskers indicate minimum and maximum values, with the line drawn at the median. Individual datapoints show the spread (n = 6 for each sequence; total n = 18). Extraction efficiencies were compared to the no carrier control sample, and significance was calculated using a linear mixed effects model. **** indicates p
    Figure Legend Snippet: MiRNA recovery with 5 μg glycogen or linear acrylamide as a co-precipitant during RNA extraction. A . The effect of glycogen or linear acrylamide (LA) on miRNA recovery was determined for the RNeasy and miRNeasy kits. Boxplot whiskers indicate minimum and maximum values, with the line drawn at the median. Individual datapoints show the spread (n = 6 for each sequence; total n = 18). Extraction efficiencies were compared to the no carrier control sample, and significance was calculated using a linear mixed effects model. **** indicates p

    Techniques Used: RNA Extraction, Sequencing

    7) Product Images from "Investigating gene expression profiles of whole blood and peripheral blood mononuclear cells using multiple collection and processing methods"

    Article Title: Investigating gene expression profiles of whole blood and peripheral blood mononuclear cells using multiple collection and processing methods

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0225137

    Sample collection, processing and RNA extraction: Whole blood was collected in triplicate followed by peripheral blood mononuclear cells (PBMCs) separation in a subset of samples. The following sample collection tubes were used for the study: RNAgard ® , PAXgene ® RNA, EDTA, ACD-A, and CPT tubes. The PBMC separation was done using standard procedures for CPT tubes, and magnetic bead, LeukoLOCK ™ and LSM methods. The samples were then stored at either 4°C (Cold) or -80°C (Frozen) and shipped overnight (o/n) for follow-up RNA extraction. Next, they were treated with one or more of several different RNA extraction procedures: Biomaxi Precip Buffer/ PAXgene ® Blood miRNA, PAXgene ® Blood miRNA, TRIzol ® LS, ACK Lysing Buffer/ Qiagen miRNeasy, and TRIzol ® Reagent manufacturer’s protocol.
    Figure Legend Snippet: Sample collection, processing and RNA extraction: Whole blood was collected in triplicate followed by peripheral blood mononuclear cells (PBMCs) separation in a subset of samples. The following sample collection tubes were used for the study: RNAgard ® , PAXgene ® RNA, EDTA, ACD-A, and CPT tubes. The PBMC separation was done using standard procedures for CPT tubes, and magnetic bead, LeukoLOCK ™ and LSM methods. The samples were then stored at either 4°C (Cold) or -80°C (Frozen) and shipped overnight (o/n) for follow-up RNA extraction. Next, they were treated with one or more of several different RNA extraction procedures: Biomaxi Precip Buffer/ PAXgene ® Blood miRNA, PAXgene ® Blood miRNA, TRIzol ® LS, ACK Lysing Buffer/ Qiagen miRNeasy, and TRIzol ® Reagent manufacturer’s protocol.

    Techniques Used: RNA Extraction, Cycling Probe Technology

    8) Product Images from "MicroRNA-205 Directly Regulates the Tumor Suppressor, Interleukin-24, in Human KB Oral Cancer Cells"

    Article Title: MicroRNA-205 Directly Regulates the Tumor Suppressor, Interleukin-24, in Human KB Oral Cancer Cells

    Journal: Molecules and Cells

    doi: 10.1007/s10059-013-2154-7

    MicroRNA array in KB cells compared with NHOK cells. Total RNA from both KB cells and NHOK were isolated with miRNeasy mini kit following the manufacturer’s instructions. The concentration, purity, and amount of total RNA were quantified using
    Figure Legend Snippet: MicroRNA array in KB cells compared with NHOK cells. Total RNA from both KB cells and NHOK were isolated with miRNeasy mini kit following the manufacturer’s instructions. The concentration, purity, and amount of total RNA were quantified using

    Techniques Used: Isolation, Concentration Assay

    9) Product Images from "Comparison of methods for milk pre-processing, exosome isolation, and RNA extraction in bovine and human milk"

    Article Title: Comparison of methods for milk pre-processing, exosome isolation, and RNA extraction in bovine and human milk

    Journal: bioRxiv

    doi: 10.1101/2020.08.14.251629

    RNA yield [ng/μL], purity and quality of human milk-derived exosome pellets and supernatants isolated via ExoQuick precipitation and ultracentrifugation methods. RNA was extracted using four protocols, 1) QIAzol + miRNeasy MiniKit, 2) TRIzol LS, 3) TRIzol + RNA Clean and Concentrator Kit (RCC), and 4) TRIzol LS + RCC. RNA concentration [ng/μL] (A), 1 % TAE agarose gel electrophoresis of exosome RNA (B), RNA purity - absorbance at 260nm/280nm (C), and absorbance at 260nm/230nm (D). Data are mean ± SEM with n = 3 technical replicates/exosome isolation method /RNA extraction protocol. Data were analyzed using a three-way analysis of variance with a Tukey post-hoc test (p ≤ 0.05). Main effect of fractionation: * (p ≤ 0.05). Main effect of exosome isolation method: $ (p ≤ 0.05). Main effect of RNA extraction protocol: # (p ≤ 0.05). Fractionation/exosome isolati on interaction: *$ (p ≤ 0.05). Fractionation/RNA extraction interaction: *# (p ≤ 0.05). Exosome isolation/RNA extraction interaction: $# (p ≤ 0.05). p-values on top of supernatant bars indicate significant difference between pellets and the corresponding supernatants of that particular exosome isolation protocol and RNA extraction protocol.
    Figure Legend Snippet: RNA yield [ng/μL], purity and quality of human milk-derived exosome pellets and supernatants isolated via ExoQuick precipitation and ultracentrifugation methods. RNA was extracted using four protocols, 1) QIAzol + miRNeasy MiniKit, 2) TRIzol LS, 3) TRIzol + RNA Clean and Concentrator Kit (RCC), and 4) TRIzol LS + RCC. RNA concentration [ng/μL] (A), 1 % TAE agarose gel electrophoresis of exosome RNA (B), RNA purity - absorbance at 260nm/280nm (C), and absorbance at 260nm/230nm (D). Data are mean ± SEM with n = 3 technical replicates/exosome isolation method /RNA extraction protocol. Data were analyzed using a three-way analysis of variance with a Tukey post-hoc test (p ≤ 0.05). Main effect of fractionation: * (p ≤ 0.05). Main effect of exosome isolation method: $ (p ≤ 0.05). Main effect of RNA extraction protocol: # (p ≤ 0.05). Fractionation/exosome isolati on interaction: *$ (p ≤ 0.05). Fractionation/RNA extraction interaction: *# (p ≤ 0.05). Exosome isolation/RNA extraction interaction: $# (p ≤ 0.05). p-values on top of supernatant bars indicate significant difference between pellets and the corresponding supernatants of that particular exosome isolation protocol and RNA extraction protocol.

    Techniques Used: Derivative Assay, Isolation, Concentration Assay, Agarose Gel Electrophoresis, RNA Extraction, Fractionation

    RNA yield [ng/μL], purity and quality of bovine milk-derived exosome pellets and supernatants isolated via ExoQuick precipitation and ultracentrifugation methods. RNA was extracted using four protocols, 1) QIAzol + miRNeasy MiniKit, 2) TRIzol LS, 3) TRIzol + RNA Clean and Concentrator Kit (RCC), and 4) TRIzol LS + RCC. RNA concentration [ng/μL] (A), 1 % TAE agarose gel electrophoresis (B), RNA purity - absorbance at 260nm/280nm (C), and absorbance at 260nm/230nm (D). Data are mean ± SEM with n = 6 independent trials/group. Data were analyzed using a three-way analysis of variance with a Tukey post-hoc test (p ≤ 0.05). Main effect of fractionation: * (p ≤ 0.05). Main effect of exosome isolation method: $ (p ≤ 0.05). Main effect of RNA extraction protocol: # (p ≤ 0.05). Fractionation/exosome isolation interaction: *$ (p ≤ 0.05). Fractionation/RNA extraction interaction: *# (p ≤ 0.05). Exosome isolation/RNA extraction interaction: $# (p ≤ 0.05). p-values on top of supernatant bars indicate significant difference between pellets and the corresponding supernatants of that particular exosome isolation protocol and RNA extraction protocol.
    Figure Legend Snippet: RNA yield [ng/μL], purity and quality of bovine milk-derived exosome pellets and supernatants isolated via ExoQuick precipitation and ultracentrifugation methods. RNA was extracted using four protocols, 1) QIAzol + miRNeasy MiniKit, 2) TRIzol LS, 3) TRIzol + RNA Clean and Concentrator Kit (RCC), and 4) TRIzol LS + RCC. RNA concentration [ng/μL] (A), 1 % TAE agarose gel electrophoresis (B), RNA purity - absorbance at 260nm/280nm (C), and absorbance at 260nm/230nm (D). Data are mean ± SEM with n = 6 independent trials/group. Data were analyzed using a three-way analysis of variance with a Tukey post-hoc test (p ≤ 0.05). Main effect of fractionation: * (p ≤ 0.05). Main effect of exosome isolation method: $ (p ≤ 0.05). Main effect of RNA extraction protocol: # (p ≤ 0.05). Fractionation/exosome isolation interaction: *$ (p ≤ 0.05). Fractionation/RNA extraction interaction: *# (p ≤ 0.05). Exosome isolation/RNA extraction interaction: $# (p ≤ 0.05). p-values on top of supernatant bars indicate significant difference between pellets and the corresponding supernatants of that particular exosome isolation protocol and RNA extraction protocol.

    Techniques Used: Derivative Assay, Isolation, Concentration Assay, Agarose Gel Electrophoresis, Fractionation, RNA Extraction

    Protein concentration [μg/mL] of human milk-derived exosome pellets and supernatants isolated via ExoQuick precipitation and ultracentrifugation methods. Total soluble proteins were extracted from the lower organic phase resulting from four RNA extraction protocols, 1) QIAzol + miRNeasy MiniKit, 2) TRIzol LS, 3) TRIzol + RNA Clean and Concentrator Kit (RCC), and 4) TRIzol LS + RCC. Data are mean ± SEM with n = 6 independent trial/group Data were analyzed using a three-way analysis of variance with a Tukey post-hoc test (p ≤ 0.05). Main effect of fractionation: * (p ≤ 0.05). Main effect of exosome isolation method: $ (p ≤ 0.05). Main effect of RNA extraction protocol: # (p ≤ 0.05). p-values on top of supernatant bars indicate significant difference between pellets and the corresponding supernatants of that particular exosome isolation protocol and RNA extraction protocol.
    Figure Legend Snippet: Protein concentration [μg/mL] of human milk-derived exosome pellets and supernatants isolated via ExoQuick precipitation and ultracentrifugation methods. Total soluble proteins were extracted from the lower organic phase resulting from four RNA extraction protocols, 1) QIAzol + miRNeasy MiniKit, 2) TRIzol LS, 3) TRIzol + RNA Clean and Concentrator Kit (RCC), and 4) TRIzol LS + RCC. Data are mean ± SEM with n = 6 independent trial/group Data were analyzed using a three-way analysis of variance with a Tukey post-hoc test (p ≤ 0.05). Main effect of fractionation: * (p ≤ 0.05). Main effect of exosome isolation method: $ (p ≤ 0.05). Main effect of RNA extraction protocol: # (p ≤ 0.05). p-values on top of supernatant bars indicate significant difference between pellets and the corresponding supernatants of that particular exosome isolation protocol and RNA extraction protocol.

    Techniques Used: Protein Concentration, Derivative Assay, Isolation, RNA Extraction, Fractionation

    Protein concentration [μg/mL] of bovine milk-derived exosome pellets and supernatants isolated via ExoQuick precipitation and ultracentrifugation methods. Total soluble proteins were extracted from the lower organic phase resulting from four RNA extraction protocols, 1) QIAzol + miRNeasy MiniKit, 2) TRIzol LS, 3) TRIzol + RNA Clean and Concentrator Kit (RCC), and 4) TRIzol LS + RCC. Data are mean ± SEM with n = 6 independent trial/group. Data were analyzed using a three-way analysis of variance with a Tukey post-hoc test (p ≤ 0.05). Main effect of fractionation: * (p ≤ 0.05). Fractionation/exosome isolation interaction: *$ (p ≤ 0.05). Fractionation/RNA extraction interaction: *# (p ≤ 0.05). p-values on top of supernatant bars indicate significant difference between pellets and the corresponding supernatants of that particular exosome isolation protocol and RNA extraction protocol.
    Figure Legend Snippet: Protein concentration [μg/mL] of bovine milk-derived exosome pellets and supernatants isolated via ExoQuick precipitation and ultracentrifugation methods. Total soluble proteins were extracted from the lower organic phase resulting from four RNA extraction protocols, 1) QIAzol + miRNeasy MiniKit, 2) TRIzol LS, 3) TRIzol + RNA Clean and Concentrator Kit (RCC), and 4) TRIzol LS + RCC. Data are mean ± SEM with n = 6 independent trial/group. Data were analyzed using a three-way analysis of variance with a Tukey post-hoc test (p ≤ 0.05). Main effect of fractionation: * (p ≤ 0.05). Fractionation/exosome isolation interaction: *$ (p ≤ 0.05). Fractionation/RNA extraction interaction: *# (p ≤ 0.05). p-values on top of supernatant bars indicate significant difference between pellets and the corresponding supernatants of that particular exosome isolation protocol and RNA extraction protocol.

    Techniques Used: Protein Concentration, Derivative Assay, Isolation, RNA Extraction, Fractionation

    10) Product Images from "Comparison of protocols and RNA carriers for plasma miRNA isolation. Unraveling RNA carrier influence on miRNA isolation"

    Article Title: Comparison of protocols and RNA carriers for plasma miRNA isolation. Unraveling RNA carrier influence on miRNA isolation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0187005

    Correlations between mean “fold recovery” (FR) values of miRNAs and base 10 logarithm of miRNA relative abundance in sample (SRA), using yE protocol (A) and yQ protocol (B). yE, miRCURY RNA isolation kit Biofluids modified protocol using yeast RNA carrier; yQ, miRNeasy Mini kit modified protocol using yeast RNA carrier; FR, the fold recovery of each miRNA vs the same protocol without carrier; SRA, the mean relative abundance of each miRNA on samples isolated without carrier.
    Figure Legend Snippet: Correlations between mean “fold recovery” (FR) values of miRNAs and base 10 logarithm of miRNA relative abundance in sample (SRA), using yE protocol (A) and yQ protocol (B). yE, miRCURY RNA isolation kit Biofluids modified protocol using yeast RNA carrier; yQ, miRNeasy Mini kit modified protocol using yeast RNA carrier; FR, the fold recovery of each miRNA vs the same protocol without carrier; SRA, the mean relative abundance of each miRNA on samples isolated without carrier.

    Techniques Used: Isolation, Modification

    Raw Cq values. Evaluation of Cq values of UniSp2, miR-103a-3p and miR-451a in four samples using different isolation protocols and RNA carriers. y, yeast RNA carrier; m, MS2 RNA carrier; w, without carrier; Q, miRNeasy Mini kit modified protocol; E, miRCURY RNA isolation kit Biofluids modified protocol. Mean and standard deviation values are indicated under each box plot diagram.
    Figure Legend Snippet: Raw Cq values. Evaluation of Cq values of UniSp2, miR-103a-3p and miR-451a in four samples using different isolation protocols and RNA carriers. y, yeast RNA carrier; m, MS2 RNA carrier; w, without carrier; Q, miRNeasy Mini kit modified protocol; E, miRCURY RNA isolation kit Biofluids modified protocol. Mean and standard deviation values are indicated under each box plot diagram.

    Techniques Used: Isolation, Modification, Standard Deviation

    Correlations between mean “fold recovery” (FR) values of miRNAs and ΔG of each miRNA, using yE protocol (A) and yQ protocol (B). To avoid the influence of GC content in the analysis of ΔG, for these correlations we used only miRNAs with a GC content between 25% and 75% percentile. yE, miRCURY RNA isolation kit Biofluids modified protocol using yeast RNA carrier; yQ, miRNeasy Mini kit modified protocol using yeast RNA carrier; FR, the fold recovery of each miRNA vs the same protocol without carrier; ΔG, the free energy of the most stable secondary structure of each miRNA.
    Figure Legend Snippet: Correlations between mean “fold recovery” (FR) values of miRNAs and ΔG of each miRNA, using yE protocol (A) and yQ protocol (B). To avoid the influence of GC content in the analysis of ΔG, for these correlations we used only miRNAs with a GC content between 25% and 75% percentile. yE, miRCURY RNA isolation kit Biofluids modified protocol using yeast RNA carrier; yQ, miRNeasy Mini kit modified protocol using yeast RNA carrier; FR, the fold recovery of each miRNA vs the same protocol without carrier; ΔG, the free energy of the most stable secondary structure of each miRNA.

    Techniques Used: Isolation, Modification

    Correlations between mean “fold recovery” (FR) values of miRNAs and GC content of each miRNA, using yE protocol (A) and yQ protocol (B). yE, miRCURY RNA isolation kit Biofluids modified protocol using yeast RNA carrier; yQ, miRNeasy Mini kit modified protocol using yeast RNA carrier; FR, the fold recovery of each miRNA vs the same protocol without carrier.
    Figure Legend Snippet: Correlations between mean “fold recovery” (FR) values of miRNAs and GC content of each miRNA, using yE protocol (A) and yQ protocol (B). yE, miRCURY RNA isolation kit Biofluids modified protocol using yeast RNA carrier; yQ, miRNeasy Mini kit modified protocol using yeast RNA carrier; FR, the fold recovery of each miRNA vs the same protocol without carrier.

    Techniques Used: Isolation, Modification

    miRNA levels normalized by plasma volume and by an endogenous miRNA. Fold abundance of miRNAs normalized by plasma volume (FR of miRNAs, left column) and normalized by hsa-miR-103a-3p (apFC of miRNAs, right column) and referred to control plasma samples isolated with the E protocol without carrier (in gray). Data were determined in plasma RNA-derived samples isolated with Q and E modified protocols with yeast RNA as carrier (y) or without RNA carrier (w). E, miRCURY RNA isolation kit Biofluids modified protocol; Q, miRNeasy Mini kit modified protocol; FR, fold recovery of miRNAs vs wE protocol; apFC, apparent fold change of miRNAs vs wE protocol. P -values were calculated using Wilcoxon Signed Rank Test, Exact Signification two-tailed, with the IBM SPSS Statistics 20 software. ϕ P
    Figure Legend Snippet: miRNA levels normalized by plasma volume and by an endogenous miRNA. Fold abundance of miRNAs normalized by plasma volume (FR of miRNAs, left column) and normalized by hsa-miR-103a-3p (apFC of miRNAs, right column) and referred to control plasma samples isolated with the E protocol without carrier (in gray). Data were determined in plasma RNA-derived samples isolated with Q and E modified protocols with yeast RNA as carrier (y) or without RNA carrier (w). E, miRCURY RNA isolation kit Biofluids modified protocol; Q, miRNeasy Mini kit modified protocol; FR, fold recovery of miRNAs vs wE protocol; apFC, apparent fold change of miRNAs vs wE protocol. P -values were calculated using Wilcoxon Signed Rank Test, Exact Signification two-tailed, with the IBM SPSS Statistics 20 software. ϕ P

    Techniques Used: Isolation, Derivative Assay, Modification, Two Tailed Test, Software

    11) Product Images from "Bias in recent miRBase annotations potentially associated with RNA quality issues"

    Article Title: Bias in recent miRBase annotations potentially associated with RNA quality issues

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-05070-0

    Experimental design. ( a ) Liver, heart and brain of male mice were harvested immediately after death, divided into 8 parts of about equal size, and stored at either 4 °C or at room temperature (RT) for the indicated time periods before RNA isolation. Experiments were performed in biological triplicates. RNA integrity was measured with Bioanalyzer. Gel-like image of brain tissue is given as example. MiRNA expression profiles of one replicate were measured using microarrays. ( b ) Liver tissue of 3 male mice was harvested immediately after death and divided into 5 parts of about equal size. Three parts were immediately transferred into RNAlater (0 h), two parts were stored for 96 h at room temperature (96 h). Two samples (0 h and 96 h) were isolated using standard procedure with miRNeasy Kit without DNase digestion. Two samples (0 h and 96 h) were isolated with optional DNase digestion to exclude DNA background. From the remaining undegraded sample (0 h), total RNA without small RNAs was isolated using RNeasy Kit with optional DNase digestion. Isolated RNA was further treated with 0 U, 0.026 U and 0.67 U RNase for 30 min to generate artificial RNA degradation. RNA integrity was measured with Bioanalyzer. MiRNA expression profiles of all replicates were measured using microarrays. The schematic drawings were prepared using the Biomedical-PPT-Toolkit-Suite from Motifolio Inc., USA.
    Figure Legend Snippet: Experimental design. ( a ) Liver, heart and brain of male mice were harvested immediately after death, divided into 8 parts of about equal size, and stored at either 4 °C or at room temperature (RT) for the indicated time periods before RNA isolation. Experiments were performed in biological triplicates. RNA integrity was measured with Bioanalyzer. Gel-like image of brain tissue is given as example. MiRNA expression profiles of one replicate were measured using microarrays. ( b ) Liver tissue of 3 male mice was harvested immediately after death and divided into 5 parts of about equal size. Three parts were immediately transferred into RNAlater (0 h), two parts were stored for 96 h at room temperature (96 h). Two samples (0 h and 96 h) were isolated using standard procedure with miRNeasy Kit without DNase digestion. Two samples (0 h and 96 h) were isolated with optional DNase digestion to exclude DNA background. From the remaining undegraded sample (0 h), total RNA without small RNAs was isolated using RNeasy Kit with optional DNase digestion. Isolated RNA was further treated with 0 U, 0.026 U and 0.67 U RNase for 30 min to generate artificial RNA degradation. RNA integrity was measured with Bioanalyzer. MiRNA expression profiles of all replicates were measured using microarrays. The schematic drawings were prepared using the Biomedical-PPT-Toolkit-Suite from Motifolio Inc., USA.

    Techniques Used: Mouse Assay, Isolation, Expressing

    12) Product Images from "Assessing cellular and circulating miRNA recovery: the impact of the RNA isolation method and the quantity of input material"

    Article Title: Assessing cellular and circulating miRNA recovery: the impact of the RNA isolation method and the quantity of input material

    Journal: Scientific Reports

    doi: 10.1038/srep19529

    Efficiency of RNA extraction methods for miRNA detection by RT-qPCR in different cell density conditions, using a fixed RNA quantity. The results represent average Cq values obtained for ( a ) mir-106a, ( b ) mir-222 and ( c ) U6 snRNA. RNA samples from 25 × 10 3 , 200 × 10 3 and 800 × 10 3 A549 cells were obtained by extraction with either Trizol ® LS, miRNeasy ® , or miRCURY™, in the presence or absence of MS2 carrier. The detection of miRNA was performed by RT-qPCR starting with 5 ng of total RNA/RT reaction. The mean values ± SD of 3 independent experiments are shown. * P
    Figure Legend Snippet: Efficiency of RNA extraction methods for miRNA detection by RT-qPCR in different cell density conditions, using a fixed RNA quantity. The results represent average Cq values obtained for ( a ) mir-106a, ( b ) mir-222 and ( c ) U6 snRNA. RNA samples from 25 × 10 3 , 200 × 10 3 and 800 × 10 3 A549 cells were obtained by extraction with either Trizol ® LS, miRNeasy ® , or miRCURY™, in the presence or absence of MS2 carrier. The detection of miRNA was performed by RT-qPCR starting with 5 ng of total RNA/RT reaction. The mean values ± SD of 3 independent experiments are shown. * P

    Techniques Used: RNA Extraction, Quantitative RT-PCR

    Efficiency of RNA extraction methods for miRNA detection by RT-qPCR in different cell density conditions, using fixed RNA volumes. RNA samples from ( a,b,c,f ) 25 × 10 3 , 200 × 10 3 and 800 × 10 3 A549 cells (n = 3) and ( d,e,g ) 100, 1000 and 10 × 10 3 A549 cells (n = 3) were obtained by extraction with either Trizol ® LS, miRNeasy ® , or miRCURY™, in the presence or absence of MS2 carrier. The results represent average Cq values obtained for ( a,d ) mir-106a, ( b,e ) mir-222, ( c ) mir-141 and ( f,g ) U6 snRNA. The detection of miRNA was performed by RT-qPCR using a fixed volume of RNA samples (see Methods for details). The mean values ± SD of 3 independent experiments are shown. * P
    Figure Legend Snippet: Efficiency of RNA extraction methods for miRNA detection by RT-qPCR in different cell density conditions, using fixed RNA volumes. RNA samples from ( a,b,c,f ) 25 × 10 3 , 200 × 10 3 and 800 × 10 3 A549 cells (n = 3) and ( d,e,g ) 100, 1000 and 10 × 10 3 A549 cells (n = 3) were obtained by extraction with either Trizol ® LS, miRNeasy ® , or miRCURY™, in the presence or absence of MS2 carrier. The results represent average Cq values obtained for ( a,d ) mir-106a, ( b,e ) mir-222, ( c ) mir-141 and ( f,g ) U6 snRNA. The detection of miRNA was performed by RT-qPCR using a fixed volume of RNA samples (see Methods for details). The mean values ± SD of 3 independent experiments are shown. * P

    Techniques Used: RNA Extraction, Quantitative RT-PCR

    Detection of microRNAs in bodily fluids by RT-qPCR, using different RNA extraction protocols and fixed RNA volumes. ( a ) RNA was extracted from 200 μL of plasma (n = 6) using either miRCURY™, miRCURY™ biofluids or miRNeasy ® protocol. Exosomes were isolated from 2 × 2 mL of plasma (n = 3), then subjected to RNA isolation by either miRNeasy ® or miRCURY™ kit. The results represent mean Cq values ± SD obtained for mir-106a, mir-222, mir-16 and mir-223, using 2.5 μL of RNA/RT reaction. Expression levels of plasma mir-106a, mir-222 and mir-223 were normalized to mir-16 levels and expressed as fold change relative to miRNeasy ® condition under the histograms. ( b ) RNA was isolated from 200 μL of plasma (n = 3) containing 25 fmol of cel-mir-39-3p as a spike-in control directly added into the lysis solution before mixing it with the plasma sample. RNA was isolated with either miRCURY™, miRCURY™ biofluids or miRNeasy ® protocols. The results represent mean Cq values ± SD obtained for the exogenous cel-mir-39, using 2.5 μL of RNA/RT reaction. ( c ) 50 to 100 mL of urine were used for urinary exosome isolation in two equal fractions. RNA was then extracted by either miRNeasy ® or miRCURY™ kit. The results represent the Cq values obtained for mir-30c-2-3p, mir-106a, mir-204, mir-222 and mir-141, generated from 3 different urinary exosome samples, using 2.5 μL of RNA/RT reaction. ** P
    Figure Legend Snippet: Detection of microRNAs in bodily fluids by RT-qPCR, using different RNA extraction protocols and fixed RNA volumes. ( a ) RNA was extracted from 200 μL of plasma (n = 6) using either miRCURY™, miRCURY™ biofluids or miRNeasy ® protocol. Exosomes were isolated from 2 × 2 mL of plasma (n = 3), then subjected to RNA isolation by either miRNeasy ® or miRCURY™ kit. The results represent mean Cq values ± SD obtained for mir-106a, mir-222, mir-16 and mir-223, using 2.5 μL of RNA/RT reaction. Expression levels of plasma mir-106a, mir-222 and mir-223 were normalized to mir-16 levels and expressed as fold change relative to miRNeasy ® condition under the histograms. ( b ) RNA was isolated from 200 μL of plasma (n = 3) containing 25 fmol of cel-mir-39-3p as a spike-in control directly added into the lysis solution before mixing it with the plasma sample. RNA was isolated with either miRCURY™, miRCURY™ biofluids or miRNeasy ® protocols. The results represent mean Cq values ± SD obtained for the exogenous cel-mir-39, using 2.5 μL of RNA/RT reaction. ( c ) 50 to 100 mL of urine were used for urinary exosome isolation in two equal fractions. RNA was then extracted by either miRNeasy ® or miRCURY™ kit. The results represent the Cq values obtained for mir-30c-2-3p, mir-106a, mir-204, mir-222 and mir-141, generated from 3 different urinary exosome samples, using 2.5 μL of RNA/RT reaction. ** P

    Techniques Used: Quantitative RT-PCR, RNA Extraction, Isolation, Expressing, Lysis, Generated

    Impact of the RNA extraction method on miRNA profiling in plasma and bodily fluid-derived exosomes, using TLDA. The Venn diagrams compare the numbers of unique and overlapping miRNAs, detected by TLDA, in RNA samples extracted with either miRNeasy ® or miRCURY™ kit. The comparison miRNeasy ® versus miRCURY™ was performed in ( a ) plasma, ( b ) plasma exosomes and ( c ) urinary exosomes. RNA extraction was done using ( a ) 200 μL of plasma, ( b ) exosomes originating from 2 mL of plasma and ( c ) exosomes isolated from 25 to 50 mL of urine. For each biological source (each panel represents data from one biological source, i.e., from plasma, plasma exosomes or urinary exosomes), two histograms display the number of miRNAs detected at high (Cq
    Figure Legend Snippet: Impact of the RNA extraction method on miRNA profiling in plasma and bodily fluid-derived exosomes, using TLDA. The Venn diagrams compare the numbers of unique and overlapping miRNAs, detected by TLDA, in RNA samples extracted with either miRNeasy ® or miRCURY™ kit. The comparison miRNeasy ® versus miRCURY™ was performed in ( a ) plasma, ( b ) plasma exosomes and ( c ) urinary exosomes. RNA extraction was done using ( a ) 200 μL of plasma, ( b ) exosomes originating from 2 mL of plasma and ( c ) exosomes isolated from 25 to 50 mL of urine. For each biological source (each panel represents data from one biological source, i.e., from plasma, plasma exosomes or urinary exosomes), two histograms display the number of miRNAs detected at high (Cq

    Techniques Used: RNA Extraction, Derivative Assay, TLDA Assay, Isolation

    13) Product Images from "Optimized microRNA purification from TRIzol-treated plasma"

    Article Title: Optimized microRNA purification from TRIzol-treated plasma

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1299-5

    MiRNA recovery from decreasing amounts of plasma input. The impact of plasma volume on the recovery of (A) exogenous RNA sequences and (B) endogenous miRNAs was evaluated using real-time RT-PCR. Plasma samples were diluted in water to 50 μL and processed with the QIAGEN miRNeasy Mini Kit. In (A) , three synthetic sequences of different amounts were spiked into the samples and measured in six technical replicates. Significance compared to the water-only control was determined using a linear mixed effects model, **** = p
    Figure Legend Snippet: MiRNA recovery from decreasing amounts of plasma input. The impact of plasma volume on the recovery of (A) exogenous RNA sequences and (B) endogenous miRNAs was evaluated using real-time RT-PCR. Plasma samples were diluted in water to 50 μL and processed with the QIAGEN miRNeasy Mini Kit. In (A) , three synthetic sequences of different amounts were spiked into the samples and measured in six technical replicates. Significance compared to the water-only control was determined using a linear mixed effects model, **** = p

    Techniques Used: Quantitative RT-PCR

    MiRNA recovery with 5 μg glycogen or linear acrylamide as a co-precipitant during RNA extraction. A . The effect of glycogen or linear acrylamide (LA) on miRNA recovery was determined for the RNeasy and miRNeasy kits. Boxplot whiskers indicate minimum and maximum values, with the line drawn at the median. Individual datapoints show the spread (n = 6 for each sequence; total n = 18). Extraction efficiencies were compared to the no carrier control sample, and significance was calculated using a linear mixed effects model. **** indicates p
    Figure Legend Snippet: MiRNA recovery with 5 μg glycogen or linear acrylamide as a co-precipitant during RNA extraction. A . The effect of glycogen or linear acrylamide (LA) on miRNA recovery was determined for the RNeasy and miRNeasy kits. Boxplot whiskers indicate minimum and maximum values, with the line drawn at the median. Individual datapoints show the spread (n = 6 for each sequence; total n = 18). Extraction efficiencies were compared to the no carrier control sample, and significance was calculated using a linear mixed effects model. **** indicates p

    Techniques Used: RNA Extraction, Sequencing

    14) Product Images from "miR-19a contributes to gefitinib resistance and epithelial mesenchymal transition in non-small cell lung cancer cells by targeting c-Met"

    Article Title: miR-19a contributes to gefitinib resistance and epithelial mesenchymal transition in non-small cell lung cancer cells by targeting c-Met

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-01153-0

    miR-19a expression in NSCLC patient serum samples and cell lines ( A ). Patients were treated with gefitinib and examined monthly with Computerized Tomographic Scanning (CT). Patients were sensitive to gefitinib at the initial stage and became resistant to gefitinib, which was detected by CT; ( B ) The expression of miR-19a was detected in patient serum samples. Serum was obtained from fifteen patients prior to oral gefitinib treatment (pre-resistance) and after they had developed gefitinib resistance (post-resistance). Total RNA was extracted from the serum using the miRNeasy mini kit (QIAGEN), and the miR-19a level was measured by real-time PCR (**p
    Figure Legend Snippet: miR-19a expression in NSCLC patient serum samples and cell lines ( A ). Patients were treated with gefitinib and examined monthly with Computerized Tomographic Scanning (CT). Patients were sensitive to gefitinib at the initial stage and became resistant to gefitinib, which was detected by CT; ( B ) The expression of miR-19a was detected in patient serum samples. Serum was obtained from fifteen patients prior to oral gefitinib treatment (pre-resistance) and after they had developed gefitinib resistance (post-resistance). Total RNA was extracted from the serum using the miRNeasy mini kit (QIAGEN), and the miR-19a level was measured by real-time PCR (**p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    15) Product Images from "Optimizing Preservation of Extracellular Vesicular miRNAs Derived from Clinical Cerebrospinal Fluid"

    Article Title: Optimizing Preservation of Extracellular Vesicular miRNAs Derived from Clinical Cerebrospinal Fluid

    Journal: Cancer biomarkers : section A of Disease markers

    doi: 10.3233/CBM-160609

    Effects of lyophilization on clinical CSF EV stability and EV associated miRNAs Lyophilized CSF EV samples were stored at room temperature for 7 days prior to rehydration with distilled water and then subjected to parallel analysis against non-lyophilized EVs. (A) Total number of EVs recovered as determined by NTA. (B) Representative TEM images of non-lyophilized EVs and EVs that were lyophilized and rehydrated. Scale bar represents 500nm in 5000× magnification images, and 200nm in 25000× magnification images. (C) EV size profile as determined by NTA. (D) Comparison of total EV RNA yield extracted using Qiagen miRNeasy mini kit. (E) Detection of miR-21, miR-24, miR-103 and miR-125 transcripts in EVs by qRT-PCR. Significant reductions in miRNA levels were observed in lyophilized EVs. Lyophilization was performed in duplicate and data are expressed as mean ± SEM. * indicates p
    Figure Legend Snippet: Effects of lyophilization on clinical CSF EV stability and EV associated miRNAs Lyophilized CSF EV samples were stored at room temperature for 7 days prior to rehydration with distilled water and then subjected to parallel analysis against non-lyophilized EVs. (A) Total number of EVs recovered as determined by NTA. (B) Representative TEM images of non-lyophilized EVs and EVs that were lyophilized and rehydrated. Scale bar represents 500nm in 5000× magnification images, and 200nm in 25000× magnification images. (C) EV size profile as determined by NTA. (D) Comparison of total EV RNA yield extracted using Qiagen miRNeasy mini kit. (E) Detection of miR-21, miR-24, miR-103 and miR-125 transcripts in EVs by qRT-PCR. Significant reductions in miRNA levels were observed in lyophilized EVs. Lyophilization was performed in duplicate and data are expressed as mean ± SEM. * indicates p

    Techniques Used: Transmission Electron Microscopy, Quantitative RT-PCR

    Clinical CSF EVs stored under Room Temperature (RT) and −80°C EVs isolated from CSF that were stored at room temperature for 1 and 7 days were comparable to EVs isolated from CSF stored at −80°C. (A) Total number of EVs recovered as determined by Nanoparticle tracking analysis (NTA). (B) Representative TEM images of EVs isolated from CSF stored under different conditions. Scale bar represents 500nm in 5000× magnification images, and 200nm in 25000× magnification images. (C) EV size profile as determined by NTA. (D) Comparison of total EV RNA yield extracted using Qiagen miRNeasy mini kit. (E) Detection of miR-21, miR-24, miR-103 and miR-125 transcripts in EVs by qRT-PCR. EV miRNAs appeared to be stable in CSF at room temperature for up to 7 days. Experiments were performed in triplicate and data are expressed as mean ± SEM.
    Figure Legend Snippet: Clinical CSF EVs stored under Room Temperature (RT) and −80°C EVs isolated from CSF that were stored at room temperature for 1 and 7 days were comparable to EVs isolated from CSF stored at −80°C. (A) Total number of EVs recovered as determined by Nanoparticle tracking analysis (NTA). (B) Representative TEM images of EVs isolated from CSF stored under different conditions. Scale bar represents 500nm in 5000× magnification images, and 200nm in 25000× magnification images. (C) EV size profile as determined by NTA. (D) Comparison of total EV RNA yield extracted using Qiagen miRNeasy mini kit. (E) Detection of miR-21, miR-24, miR-103 and miR-125 transcripts in EVs by qRT-PCR. EV miRNAs appeared to be stable in CSF at room temperature for up to 7 days. Experiments were performed in triplicate and data are expressed as mean ± SEM.

    Techniques Used: Isolation, Transmission Electron Microscopy, Quantitative RT-PCR

    Related Articles

    Isolation:

    Article Title: Optimized microRNA purification from TRIzol-treated plasma
    Article Snippet: .. Total RNA purification using spin column-based kits The following commercially available RNA purification kits were tested in this study: mir Vana miRNA Isolation Kit (Ambion, Austin, TX), miRCURY Isolation Kit – Biofluids (Exiqon, Inc.), RNeasy Mini Kit (QIAGEN Inc., Valencia, CA), miRNeasy Mini Kit (QIAGEN Inc.), and Direct-zol RNA MiniPrep Kit (Zymo Research Corp., Irvine, CA). .. Total RNA was purified from the aqueous phase, with the exception of the Zymo Direct-zol kit, which was used immediately after the addition of the RNA spike-ins.

    Article Title: Controlled ovarian hyperstimulation induced changes in the expression of circulatory miRNA in bovine follicular fluid and blood plasma
    Article Snippet: .. Western blot analysis Exosomal and Ago2 immunoprecipitate proteins were isolated from organic phenol part during total RNA isolation using miRNeasy mini kit (Qiagen, Hilden, Germany) and resuspended in 8 M Urea. .. Approximately 20 μg of protein from each sample were resolved in 12 % SDS-PAGE polyacrylamide gel (Bio-Rad, Corp., Hercules, CA, USA) and immune reactive proteins were visualized with a Chemidoc XRS (Bio-Rad) instrument.

    Article Title: MicroRNA-205 Directly Regulates the Tumor Suppressor, Interleukin-24, in Human KB Oral Cancer Cells
    Article Snippet: .. Total RNA from both KB cells and NHOK were isolated with miRNeasy mini kit (Qiagen, USA) following the manufacturer’s instructions. .. The concentration, purity, and amount of total RNA were quantified using the Nano-Drop® ND-1000 unltraviolet Spectrophotometer (Thermo Scientific, USA).

    Lysis:

    Article Title: Comparison of methods for milk pre-processing, exosome isolation, and RNA extraction in bovine and human milk
    Article Snippet: .. Method 1: QIAzol + miRNeasy Mini Kit QIAzol lysis reagent combined with miRNeasy Mini Kit (Qiagen; 217004) was used as per the manufacturer’s instructions with slight modifications. .. QIAzol reagent was added to 200 μL of isolated milk exosomes (5:1, v/v).

    Purification:

    Article Title: Optimized microRNA purification from TRIzol-treated plasma
    Article Snippet: .. Total RNA purification using spin column-based kits The following commercially available RNA purification kits were tested in this study: mir Vana miRNA Isolation Kit (Ambion, Austin, TX), miRCURY Isolation Kit – Biofluids (Exiqon, Inc.), RNeasy Mini Kit (QIAGEN Inc., Valencia, CA), miRNeasy Mini Kit (QIAGEN Inc.), and Direct-zol RNA MiniPrep Kit (Zymo Research Corp., Irvine, CA). .. Total RNA was purified from the aqueous phase, with the exception of the Zymo Direct-zol kit, which was used immediately after the addition of the RNA spike-ins.

    Quantitative RT-PCR:

    Article Title: LncRNA-MEG3 inhibits activation of hepatic stellate cells through SMO protein and miR-212
    Article Snippet: .. qRT-PCR miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) was used to extract the total RNA from primary cells as well as liver tissues. .. Then, ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) was used to reverse-transcribed total RNA to cDNA.

    Article Title: lncRNA TUG1 Promotes Cell Proliferation, Migration, and Invasion in Hepatocellular Carcinoma via Regulating miR-29c-3p/COL1A1 Axis
    Article Snippet: .. qRT-PCR MiRNeasy Mini Kit (Qiagen) was used for the extraction of total RNA from tissue samples and cells. .. Reverse transcription was carried out using the PrimeScriptTM RT Master Mix (Takara, Dalian, China).

    Western Blot:

    Article Title: Controlled ovarian hyperstimulation induced changes in the expression of circulatory miRNA in bovine follicular fluid and blood plasma
    Article Snippet: .. Western blot analysis Exosomal and Ago2 immunoprecipitate proteins were isolated from organic phenol part during total RNA isolation using miRNeasy mini kit (Qiagen, Hilden, Germany) and resuspended in 8 M Urea. .. Approximately 20 μg of protein from each sample were resolved in 12 % SDS-PAGE polyacrylamide gel (Bio-Rad, Corp., Hercules, CA, USA) and immune reactive proteins were visualized with a Chemidoc XRS (Bio-Rad) instrument.

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    Qiagen qrt pcr mirneasy mini kit
    MEG3 physically interacts with SMO protein. a The overall interaction propensity of MEG3 and SMO protein was predicted by catRAPID. b Predicted interaction between MEG3 (nucleotide positions 0–1200 nt) and SMO protein (amino acid residues 0–720). c RIP experiments were performed using SMO antibody in primary HSCs at Day 0. <t>qRT-PCR</t> was performed to detect pulled-down MEG3. hnRNP-K antibody and IgG were used as positive and negative controls, respectively. d SMO asscociated MEG3 was detected by regular RT-PCR. e Mapping the SMO interaction region of MEG3. Biotinylated RNAs corresponding to different fragments of MEG3 or its antisense sequences (red line) were co-incubated with cell lysates and associated SMO proteins were detected by immunoblotting
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    MEG3 physically interacts with SMO protein. a The overall interaction propensity of MEG3 and SMO protein was predicted by catRAPID. b Predicted interaction between MEG3 (nucleotide positions 0–1200 nt) and SMO protein (amino acid residues 0–720). c RIP experiments were performed using SMO antibody in primary HSCs at Day 0. qRT-PCR was performed to detect pulled-down MEG3. hnRNP-K antibody and IgG were used as positive and negative controls, respectively. d SMO asscociated MEG3 was detected by regular RT-PCR. e Mapping the SMO interaction region of MEG3. Biotinylated RNAs corresponding to different fragments of MEG3 or its antisense sequences (red line) were co-incubated with cell lysates and associated SMO proteins were detected by immunoblotting

    Journal: Cell Death & Disease

    Article Title: LncRNA-MEG3 inhibits activation of hepatic stellate cells through SMO protein and miR-212

    doi: 10.1038/s41419-018-1068-x

    Figure Lengend Snippet: MEG3 physically interacts with SMO protein. a The overall interaction propensity of MEG3 and SMO protein was predicted by catRAPID. b Predicted interaction between MEG3 (nucleotide positions 0–1200 nt) and SMO protein (amino acid residues 0–720). c RIP experiments were performed using SMO antibody in primary HSCs at Day 0. qRT-PCR was performed to detect pulled-down MEG3. hnRNP-K antibody and IgG were used as positive and negative controls, respectively. d SMO asscociated MEG3 was detected by regular RT-PCR. e Mapping the SMO interaction region of MEG3. Biotinylated RNAs corresponding to different fragments of MEG3 or its antisense sequences (red line) were co-incubated with cell lysates and associated SMO proteins were detected by immunoblotting

    Article Snippet: qRT-PCR miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) was used to extract the total RNA from primary cells as well as liver tissues.

    Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Incubation

    Downregulation of MEG3 in liver fibrosis. a Collagen and α-SMA were analyzed in CCl 4 mice by Masson staining and immunohistochemistry, respectively. Scale bar, 100 μm. b MEG3-1, MEG3-2, and MEG3-3 expressions were detected by qRT-PCR in CCl 4 mice. c Expressions of MEG3-1, MEG3-2, and MEG3-3 were analyzed in primary HSCs at Day 0 and Day 4. Primary HSCs were isolated from the livers of healthy mice. d MEG3 was analyzed in primary HSCs isolated from oil- or CCl 4 -treated mice. e MEG3 was analyzed in primary HSCs and primary hepatocytes from the livers of healthy mice. * P

    Journal: Cell Death & Disease

    Article Title: LncRNA-MEG3 inhibits activation of hepatic stellate cells through SMO protein and miR-212

    doi: 10.1038/s41419-018-1068-x

    Figure Lengend Snippet: Downregulation of MEG3 in liver fibrosis. a Collagen and α-SMA were analyzed in CCl 4 mice by Masson staining and immunohistochemistry, respectively. Scale bar, 100 μm. b MEG3-1, MEG3-2, and MEG3-3 expressions were detected by qRT-PCR in CCl 4 mice. c Expressions of MEG3-1, MEG3-2, and MEG3-3 were analyzed in primary HSCs at Day 0 and Day 4. Primary HSCs were isolated from the livers of healthy mice. d MEG3 was analyzed in primary HSCs isolated from oil- or CCl 4 -treated mice. e MEG3 was analyzed in primary HSCs and primary hepatocytes from the livers of healthy mice. * P

    Article Snippet: qRT-PCR miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) was used to extract the total RNA from primary cells as well as liver tissues.

    Techniques: Mouse Assay, Staining, Immunohistochemistry, Quantitative RT-PCR, Isolation

    The expression of taurine upregulated gene 1 (TUG1) in primary hepatocellular carcinoma (HCC) tissues and cell lines was detected by quantitative reverse-transcription PCR (qRT-PCR). ( A ) The expression of TUG1 in 51 paired HCC tissues and matched adjacent non-tumor tissues was examined by qRT-PCR. P

    Journal: Cancer Management and Research

    Article Title: lncRNA TUG1 Promotes Cell Proliferation, Migration, and Invasion in Hepatocellular Carcinoma via Regulating miR-29c-3p/COL1A1 Axis

    doi: 10.2147/CMAR.S256624

    Figure Lengend Snippet: The expression of taurine upregulated gene 1 (TUG1) in primary hepatocellular carcinoma (HCC) tissues and cell lines was detected by quantitative reverse-transcription PCR (qRT-PCR). ( A ) The expression of TUG1 in 51 paired HCC tissues and matched adjacent non-tumor tissues was examined by qRT-PCR. P

    Article Snippet: qRT-PCR MiRNeasy Mini Kit (Qiagen) was used for the extraction of total RNA from tissue samples and cells.

    Techniques: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR

    Taurine upregulated gene 1 (TUG1) knockdown inhibited the proliferation, migration and invasion of hepatocellular carcinoma (HCC) cells. HeP3B and sk-Hep-1 cells were transfected with si-TUG-1, si-TUG-2 or si-NC. ( A ) qRT-PCR was used to detect the expression of TUG1. ( B ) 3-(4,5-Dimethyl-2-Thiazolyl)-2, 5-Diphenyl-2-H-Tetrazolium Bromide (MTT) assay was applied to measure cell proliferation at 0, 24, 48, 72 and 96 h in transfected HeP3B and sk-Hep-1 cells. ( C ) Wound-healing assay was conducted to evaluate the migration of transfected HeP3B and sk-Hep-1 cells. ( D ) Transwell invasion assay was used to detect the invasion of transfected HeP3B and sk-Hep-1 cells. ** P

    Journal: Cancer Management and Research

    Article Title: lncRNA TUG1 Promotes Cell Proliferation, Migration, and Invasion in Hepatocellular Carcinoma via Regulating miR-29c-3p/COL1A1 Axis

    doi: 10.2147/CMAR.S256624

    Figure Lengend Snippet: Taurine upregulated gene 1 (TUG1) knockdown inhibited the proliferation, migration and invasion of hepatocellular carcinoma (HCC) cells. HeP3B and sk-Hep-1 cells were transfected with si-TUG-1, si-TUG-2 or si-NC. ( A ) qRT-PCR was used to detect the expression of TUG1. ( B ) 3-(4,5-Dimethyl-2-Thiazolyl)-2, 5-Diphenyl-2-H-Tetrazolium Bromide (MTT) assay was applied to measure cell proliferation at 0, 24, 48, 72 and 96 h in transfected HeP3B and sk-Hep-1 cells. ( C ) Wound-healing assay was conducted to evaluate the migration of transfected HeP3B and sk-Hep-1 cells. ( D ) Transwell invasion assay was used to detect the invasion of transfected HeP3B and sk-Hep-1 cells. ** P

    Article Snippet: qRT-PCR MiRNeasy Mini Kit (Qiagen) was used for the extraction of total RNA from tissue samples and cells.

    Techniques: Migration, Transfection, Quantitative RT-PCR, Expressing, MTT Assay, Wound Healing Assay, Transwell Invasion Assay

    Specificity of isolation of exosomes and Ago2 protein complex from follicular fluid and blood plasma. Exosome and Ago2 proteins were isolated from organic-phenol fraction during total RNA isolation using miRNeasy kit and resolved in 8M urea. Protein concentrations were quantified using Bradford assay and total of twenty microgram protein from each group were separated in 12 % SDS-PAGE, transferred nitrocellulose membrane and incubated with specific antibody (CD63 and Ago2). Followed by HRP-conjugated secondary antibody and detected using chemiluminescent substrate. Western-blot results indicate the specificity of exosome and Ago2 protein isolation from follicular fluid (FF) and blood plasma (BP) as indicated in the corresponding figures

    Journal: Journal of Ovarian Research

    Article Title: Controlled ovarian hyperstimulation induced changes in the expression of circulatory miRNA in bovine follicular fluid and blood plasma

    doi: 10.1186/s13048-015-0208-5

    Figure Lengend Snippet: Specificity of isolation of exosomes and Ago2 protein complex from follicular fluid and blood plasma. Exosome and Ago2 proteins were isolated from organic-phenol fraction during total RNA isolation using miRNeasy kit and resolved in 8M urea. Protein concentrations were quantified using Bradford assay and total of twenty microgram protein from each group were separated in 12 % SDS-PAGE, transferred nitrocellulose membrane and incubated with specific antibody (CD63 and Ago2). Followed by HRP-conjugated secondary antibody and detected using chemiluminescent substrate. Western-blot results indicate the specificity of exosome and Ago2 protein isolation from follicular fluid (FF) and blood plasma (BP) as indicated in the corresponding figures

    Article Snippet: Western blot analysis Exosomal and Ago2 immunoprecipitate proteins were isolated from organic phenol part during total RNA isolation using miRNeasy mini kit (Qiagen, Hilden, Germany) and resuspended in 8 M Urea.

    Techniques: Isolation, Bradford Assay, SDS Page, Incubation, Western Blot

    Experimental design. ( a ) Liver, heart and brain of male mice were harvested immediately after death, divided into 8 parts of about equal size, and stored at either 4 °C or at room temperature (RT) for the indicated time periods before RNA isolation. Experiments were performed in biological triplicates. RNA integrity was measured with Bioanalyzer. Gel-like image of brain tissue is given as example. MiRNA expression profiles of one replicate were measured using microarrays. ( b ) Liver tissue of 3 male mice was harvested immediately after death and divided into 5 parts of about equal size. Three parts were immediately transferred into RNAlater (0 h), two parts were stored for 96 h at room temperature (96 h). Two samples (0 h and 96 h) were isolated using standard procedure with miRNeasy Kit without DNase digestion. Two samples (0 h and 96 h) were isolated with optional DNase digestion to exclude DNA background. From the remaining undegraded sample (0 h), total RNA without small RNAs was isolated using RNeasy Kit with optional DNase digestion. Isolated RNA was further treated with 0 U, 0.026 U and 0.67 U RNase for 30 min to generate artificial RNA degradation. RNA integrity was measured with Bioanalyzer. MiRNA expression profiles of all replicates were measured using microarrays. The schematic drawings were prepared using the Biomedical-PPT-Toolkit-Suite from Motifolio Inc., USA.

    Journal: Scientific Reports

    Article Title: Bias in recent miRBase annotations potentially associated with RNA quality issues

    doi: 10.1038/s41598-017-05070-0

    Figure Lengend Snippet: Experimental design. ( a ) Liver, heart and brain of male mice were harvested immediately after death, divided into 8 parts of about equal size, and stored at either 4 °C or at room temperature (RT) for the indicated time periods before RNA isolation. Experiments were performed in biological triplicates. RNA integrity was measured with Bioanalyzer. Gel-like image of brain tissue is given as example. MiRNA expression profiles of one replicate were measured using microarrays. ( b ) Liver tissue of 3 male mice was harvested immediately after death and divided into 5 parts of about equal size. Three parts were immediately transferred into RNAlater (0 h), two parts were stored for 96 h at room temperature (96 h). Two samples (0 h and 96 h) were isolated using standard procedure with miRNeasy Kit without DNase digestion. Two samples (0 h and 96 h) were isolated with optional DNase digestion to exclude DNA background. From the remaining undegraded sample (0 h), total RNA without small RNAs was isolated using RNeasy Kit with optional DNase digestion. Isolated RNA was further treated with 0 U, 0.026 U and 0.67 U RNase for 30 min to generate artificial RNA degradation. RNA integrity was measured with Bioanalyzer. MiRNA expression profiles of all replicates were measured using microarrays. The schematic drawings were prepared using the Biomedical-PPT-Toolkit-Suite from Motifolio Inc., USA.

    Article Snippet: Afterwards, RNAse was inactivated with 700 µl Qiazol and RNA including any small fragments was reisolated using miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturers protocol.

    Techniques: Mouse Assay, Isolation, Expressing