trizol ls  (Thermo Fisher)


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    Name:
    TRIzol LS Reagent
    Description:
    TRIzol LS Reagent is a complete ready to use reagent optimized for the isolation of high quality total RNA or the simultaneous isolation of RNA DNA and protein from a variety of liquid samples This monophasic solution of phenol and guanidine isothiocyanate is designed to isolate separate fractions of RNA DNA and proteins from liquid samples of human animal plant yeast bacterial and viral origin typically within one hour Key features of TRIzol LS Reagent • Formulated for use with liquid samples such as serum and virus preparations• Facilitates recovery of RNA DNA and protein from a single liquid sample• Offers excellent lysis capability even with difficult biological fluidsReliably purify RNA from multiple sample sourcesTRIzol LS Reagent is designed for processing a variety of liquid samples of up to 0 25 mL in volume TRIzol LS Reagent differs from the standard TRIzol Reagent in concentration which permits larger samples to be processed Just as with the standard TRIzol Reagent the integrity of resulting RNA preparations is maintained by the highly effective inhibition of RNase activity during sample homogenization The simplicity of the TRIzol LS Reagent method allows simultaneous processing of a large number of samples The entire procedure can be completed in 1 hour Total RNA isolated by TRIzol LS Reagent is free of protein and DNA contamination Formulated to serve multiple isolationsTRIzol LS Reagent allows you to perform sequential precipitation of RNA DNA and proteins from a single sample After homogenizing the sample with TRIzol LS Reagent chloroform is added and the homogenate is allowed to separate into a clear upper aqueous layer containing RNA an interface and a red lower organic layer containing the DNA and proteins RNA is precipitated from the aqueous layer with isopropanol DNA is precipitated from the aqueous organic interface with ethanol Protein is precipitated from the phenol ethanol layer by isopropanol precipitation The precipitated RNA DNA or protein is washed to remove impurities and then resuspended for use in downstream applications Purified products are ideal for use with a variety of applicationsIsolated RNA can be used in real time quantitative PCR qPCR northern blot analysis dot blot hybridization poly A selection In vitro translation RNase protection assays and molecular cloning Isolated DNA can be used in PCR restriction enzyme digestion and Southern blots Isolated protein can be used for western blots recovery of some enzymatic activity and some immunoprecipitation
    Catalog Number:
    10296010
    Price:
    None
    Applications:
    DNA & RNA Purification & Analysis|RNAi, Epigenetics & Non-Coding RNA Research|RNA Extraction|Total RNA Isolation|Total RNA from Liquid Samples (e.g. Serum, Virus)|miRNA Isolation|miRNA & Non-Coding RNA Analysis|Viral RNA⁄DNA Purification
    Category:
    Kits and Assays
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    Structured Review

    Thermo Fisher trizol ls
    Candidate miRNA levels are elevated in EVs of cHL patients compared with healthy controls. RT-PCR analysis of miR127-3p ( A ), miR155-5p ( B ), miR21-5p ( C ), let7a-5p ( D ), miR24-3p ( E ), and miR10b-5p ( F ) in plasma extracellular vesicles (EVs) of healthy individuals ( n = 9) and cHL patients ( n = 20) after size-exclusion chromatography (SEC) and total <t>RNA</t> isolation using <t>TRIzol.</t> For each individual sample, the mean Ct value of SEC fractions 9 and 10 was used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. * P
    TRIzol LS Reagent is a complete ready to use reagent optimized for the isolation of high quality total RNA or the simultaneous isolation of RNA DNA and protein from a variety of liquid samples This monophasic solution of phenol and guanidine isothiocyanate is designed to isolate separate fractions of RNA DNA and proteins from liquid samples of human animal plant yeast bacterial and viral origin typically within one hour Key features of TRIzol LS Reagent • Formulated for use with liquid samples such as serum and virus preparations• Facilitates recovery of RNA DNA and protein from a single liquid sample• Offers excellent lysis capability even with difficult biological fluidsReliably purify RNA from multiple sample sourcesTRIzol LS Reagent is designed for processing a variety of liquid samples of up to 0 25 mL in volume TRIzol LS Reagent differs from the standard TRIzol Reagent in concentration which permits larger samples to be processed Just as with the standard TRIzol Reagent the integrity of resulting RNA preparations is maintained by the highly effective inhibition of RNase activity during sample homogenization The simplicity of the TRIzol LS Reagent method allows simultaneous processing of a large number of samples The entire procedure can be completed in 1 hour Total RNA isolated by TRIzol LS Reagent is free of protein and DNA contamination Formulated to serve multiple isolationsTRIzol LS Reagent allows you to perform sequential precipitation of RNA DNA and proteins from a single sample After homogenizing the sample with TRIzol LS Reagent chloroform is added and the homogenate is allowed to separate into a clear upper aqueous layer containing RNA an interface and a red lower organic layer containing the DNA and proteins RNA is precipitated from the aqueous layer with isopropanol DNA is precipitated from the aqueous organic interface with ethanol Protein is precipitated from the phenol ethanol layer by isopropanol precipitation The precipitated RNA DNA or protein is washed to remove impurities and then resuspended for use in downstream applications Purified products are ideal for use with a variety of applicationsIsolated RNA can be used in real time quantitative PCR qPCR northern blot analysis dot blot hybridization poly A selection In vitro translation RNase protection assays and molecular cloning Isolated DNA can be used in PCR restriction enzyme digestion and Southern blots Isolated protein can be used for western blots recovery of some enzymatic activity and some immunoprecipitation
    https://www.bioz.com/result/trizol ls/product/Thermo Fisher
    Average 99 stars, based on 440 article reviews
    Price from $9.99 to $1999.99
    trizol ls - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients"

    Article Title: Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients

    Journal: JCI Insight

    doi: 10.1172/jci.insight.89631

    Candidate miRNA levels are elevated in EVs of cHL patients compared with healthy controls. RT-PCR analysis of miR127-3p ( A ), miR155-5p ( B ), miR21-5p ( C ), let7a-5p ( D ), miR24-3p ( E ), and miR10b-5p ( F ) in plasma extracellular vesicles (EVs) of healthy individuals ( n = 9) and cHL patients ( n = 20) after size-exclusion chromatography (SEC) and total RNA isolation using TRIzol. For each individual sample, the mean Ct value of SEC fractions 9 and 10 was used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. * P
    Figure Legend Snippet: Candidate miRNA levels are elevated in EVs of cHL patients compared with healthy controls. RT-PCR analysis of miR127-3p ( A ), miR155-5p ( B ), miR21-5p ( C ), let7a-5p ( D ), miR24-3p ( E ), and miR10b-5p ( F ) in plasma extracellular vesicles (EVs) of healthy individuals ( n = 9) and cHL patients ( n = 20) after size-exclusion chromatography (SEC) and total RNA isolation using TRIzol. For each individual sample, the mean Ct value of SEC fractions 9 and 10 was used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. * P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Size-exclusion Chromatography, Isolation

    EV outperforms total plasma for monitoring treatment response and corresponds with TARC. ( A ) RT-PCR analysis of miR127-3p in total plasma of cHL patients ( n = 7) before and after treatment, after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in plasma extracellular vesicles (EVs) of the same cHL patients ( n = 7) as in A , after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ( C and D ) As in A and B , but for miR155-5p. ( E and F ) RT-PCR analysis of miR21-5p, miR155-5p, and miR127-3p in total plasma ( E ) and in plasma EVs ( F ) of an individual cHL patient with primary tumor before and after first-line treatment (gray symbols) and a cHL patient with relapsed disease before and after second-line treatment (black symbols). ( G – J ) RT-PCR analysis of miR127-3p ( G ), miR155-5p ( H ), miR21-5p ( I ), and let7a-5p ( J ) in plasma EVs of cHL patients before and after treatment ( n = 7). Each data point is the mean Ct value of the 2 consecutive SEC fractions 9 and 10. ( K ) Serum TARC levels in the same cHL patients as in G–J before and after treatment, as measured by ELISA. Data are shown as paired before and after therapy samples ( E–K ).
    Figure Legend Snippet: EV outperforms total plasma for monitoring treatment response and corresponds with TARC. ( A ) RT-PCR analysis of miR127-3p in total plasma of cHL patients ( n = 7) before and after treatment, after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in plasma extracellular vesicles (EVs) of the same cHL patients ( n = 7) as in A , after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ( C and D ) As in A and B , but for miR155-5p. ( E and F ) RT-PCR analysis of miR21-5p, miR155-5p, and miR127-3p in total plasma ( E ) and in plasma EVs ( F ) of an individual cHL patient with primary tumor before and after first-line treatment (gray symbols) and a cHL patient with relapsed disease before and after second-line treatment (black symbols). ( G – J ) RT-PCR analysis of miR127-3p ( G ), miR155-5p ( H ), miR21-5p ( I ), and let7a-5p ( J ) in plasma EVs of cHL patients before and after treatment ( n = 7). Each data point is the mean Ct value of the 2 consecutive SEC fractions 9 and 10. ( K ) Serum TARC levels in the same cHL patients as in G–J before and after treatment, as measured by ELISA. Data are shown as paired before and after therapy samples ( E–K ).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay

    Small RNA distribution and recovery in EV fractions 9 and 10. ( A and B ) RNA distribution of miR142-3p, let7a-5p, and vtRNA1-1 ( A ) and miR92a-3p, miR21-5p, and miR451-5p ( B ) in 26 fractions upon size-exclusion chromatography (SEC) of 1.5 ml healthy donor plasma. Total RNA was isolated with TRIzol followed by RT-PCR. Data are depicted as raw Ct values; error bars represent SEM from PCR duplicates. ( C ) Fold enrichment of vtRNA1-1, let7a-5p, and miR142-3p in plasma extracellular vesicles (EVs) (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of 2 donors; dots indicate individual samples. ( D ) Fold enrichment of miR92a-3p, miR21-5p, and miR451-5p in protein/HDL (fractions 20 and 21) compared with plasma EVs (fractions 9 and 10). Data are shown as the mean of 2 donors; dots indicate individual samples. ( E ) Fold enrichment of vtRNA1-1 in tumor EV (tEV; fractions 9 and 10) compared with protein/HDL (fractions 20 and 21) after SEC of 1.5 ml B cell culture supernatant. ( F ) SEC of 1.5 ml healthy donor plasma after spike in with 50 μl tumor cell line–derived exosomes. Shown is the fold increase of EBV-miR BHRF1-3 and BART2-5p in EV (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of the 2 consecutive SEC fractions; dots represent individual fractions ( E and F ).
    Figure Legend Snippet: Small RNA distribution and recovery in EV fractions 9 and 10. ( A and B ) RNA distribution of miR142-3p, let7a-5p, and vtRNA1-1 ( A ) and miR92a-3p, miR21-5p, and miR451-5p ( B ) in 26 fractions upon size-exclusion chromatography (SEC) of 1.5 ml healthy donor plasma. Total RNA was isolated with TRIzol followed by RT-PCR. Data are depicted as raw Ct values; error bars represent SEM from PCR duplicates. ( C ) Fold enrichment of vtRNA1-1, let7a-5p, and miR142-3p in plasma extracellular vesicles (EVs) (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of 2 donors; dots indicate individual samples. ( D ) Fold enrichment of miR92a-3p, miR21-5p, and miR451-5p in protein/HDL (fractions 20 and 21) compared with plasma EVs (fractions 9 and 10). Data are shown as the mean of 2 donors; dots indicate individual samples. ( E ) Fold enrichment of vtRNA1-1 in tumor EV (tEV; fractions 9 and 10) compared with protein/HDL (fractions 20 and 21) after SEC of 1.5 ml B cell culture supernatant. ( F ) SEC of 1.5 ml healthy donor plasma after spike in with 50 μl tumor cell line–derived exosomes. Shown is the fold increase of EBV-miR BHRF1-3 and BART2-5p in EV (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of the 2 consecutive SEC fractions; dots represent individual fractions ( E and F ).

    Techniques Used: Size-exclusion Chromatography, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Cell Culture, Derivative Assay

    miR127-3p EV outperforms total plasma in distinguishing cHL patients from controls. ( A ) RT-PCR analysis of miR127-3p in total plasma of healthy controls ( n = 7) and cHL patients ( n = 8) after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in extracellular vesicle (EV) fractions of the same healthy individuals and cHL patients as in A after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. ( A and B ) Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ** P
    Figure Legend Snippet: miR127-3p EV outperforms total plasma in distinguishing cHL patients from controls. ( A ) RT-PCR analysis of miR127-3p in total plasma of healthy controls ( n = 7) and cHL patients ( n = 8) after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in extracellular vesicle (EV) fractions of the same healthy individuals and cHL patients as in A after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. ( A and B ) Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ** P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Size-exclusion Chromatography

    2) Product Images from "Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses"

    Article Title: Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.01.002

    Ex-miRNA Are Stable and Protected from RNases in BALF (A) qPCR of synthetic miRNAs (“calibrators” Cal1 and Cal2) spiked into Trizol or BALF with or without an RNase inhibitor (RNAsin) (n = 3 from 1–2 independent experiments, 1-way ANOVA with Bonferroni’s multiple comparison test). (B) qPCR of analysis of miRNAs from BALF and RNA pellets treated with RNaseA (n = 3 from 3 independent experiments, 2-way ANOVA with Bonferroni’s multiple comparison test). Error bars are mean + SD; *p
    Figure Legend Snippet: Ex-miRNA Are Stable and Protected from RNases in BALF (A) qPCR of synthetic miRNAs (“calibrators” Cal1 and Cal2) spiked into Trizol or BALF with or without an RNase inhibitor (RNAsin) (n = 3 from 1–2 independent experiments, 1-way ANOVA with Bonferroni’s multiple comparison test). (B) qPCR of analysis of miRNAs from BALF and RNA pellets treated with RNaseA (n = 3 from 3 independent experiments, 2-way ANOVA with Bonferroni’s multiple comparison test). Error bars are mean + SD; *p

    Techniques Used: Real-time Polymerase Chain Reaction

    3) Product Images from "MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA"

    Article Title: MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA

    Journal: Journal of Extracellular Vesicles

    doi: 10.3402/jev.v5.29828

    (a) Western blot analysis of CTB-, AV- and ST-bound MSC EVs. MSC CM was incubated with CTB, AV or ST followed by incubation with Dynabeads conjugated with Streptavidin. The beads were immobilised with a magnet, washed, denatured and resolved onto polyacrylamide gels before electroblotting onto a nitrocellulose membrane. The membrane was probed with a primary antibody followed by horseradish peroxidase-coupled secondary antibodies against the primary antibody. The blot was then incubated with a chemiluminescent HRP substrate to detect bound primary antibody. (b) 10 µg MSC EV was extracted sequentially with biotinylated CTB and then biotinylated AV or vice versa. After each extraction, the ligand-bound vesicles were removed with Dynabeads ® MyOne Streptavidin T1 and assayed for CD81 by ELISA. The relative level of CD81 in CTB-vesicles before and after extraction with AV, and that in AV-vesicles before and after extraction with CTB were normalized to that in AV-vesicles before CTB extraction. (c) RNA analysis of CTB-, AV- and ST-EVs. CTB-, AV- or ST-binding EVs were isolated as described above and extracted for RNA using Trizol. The pellet in each of extracts was re-suspended in 50 µL of RNase-free water. 10 µL of each RNA solution was resolved on a 15% Novex Tris-borate-EDTA(TBE)-urea gel before staining with ethidium bromide.
    Figure Legend Snippet: (a) Western blot analysis of CTB-, AV- and ST-bound MSC EVs. MSC CM was incubated with CTB, AV or ST followed by incubation with Dynabeads conjugated with Streptavidin. The beads were immobilised with a magnet, washed, denatured and resolved onto polyacrylamide gels before electroblotting onto a nitrocellulose membrane. The membrane was probed with a primary antibody followed by horseradish peroxidase-coupled secondary antibodies against the primary antibody. The blot was then incubated with a chemiluminescent HRP substrate to detect bound primary antibody. (b) 10 µg MSC EV was extracted sequentially with biotinylated CTB and then biotinylated AV or vice versa. After each extraction, the ligand-bound vesicles were removed with Dynabeads ® MyOne Streptavidin T1 and assayed for CD81 by ELISA. The relative level of CD81 in CTB-vesicles before and after extraction with AV, and that in AV-vesicles before and after extraction with CTB were normalized to that in AV-vesicles before CTB extraction. (c) RNA analysis of CTB-, AV- and ST-EVs. CTB-, AV- or ST-binding EVs were isolated as described above and extracted for RNA using Trizol. The pellet in each of extracts was re-suspended in 50 µL of RNase-free water. 10 µL of each RNA solution was resolved on a 15% Novex Tris-borate-EDTA(TBE)-urea gel before staining with ethidium bromide.

    Techniques Used: Western Blot, CtB Assay, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Isolation, Staining

    4) Product Images from "Evaluation of Pre-Analytical Variables in the Quantification of Dengue Virus by Real-Time Polymerase Chain Reaction"

    Article Title: Evaluation of Pre-Analytical Variables in the Quantification of Dengue Virus by Real-Time Polymerase Chain Reaction

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.2353/jmoldx.2009.080164

    Comparison of Trizol LS and High Pure Viral RNA isolation kits in the isolation of viral RNA from DENV-3 infected patient sera. A volume of 50 μl of serum samples from dengue-infected patients were re-suspended in 150 μl of human serum
    Figure Legend Snippet: Comparison of Trizol LS and High Pure Viral RNA isolation kits in the isolation of viral RNA from DENV-3 infected patient sera. A volume of 50 μl of serum samples from dengue-infected patients were re-suspended in 150 μl of human serum

    Techniques Used: Isolation, Infection

    5) Product Images from "Circulating miRNA signatures of early pregnancy in cattle"

    Article Title: Circulating miRNA signatures of early pregnancy in cattle

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-2529-1

    Optimisation of methodology for profiling miRNAs in bovine plasma. a RT-qPCR data plots (with mean ± SEM) showing abundance of the exogenous miRNA, cel-miR-39-3p, in plasma samples after RNA extraction using 3 different kits as indicated. b RNA yield from 1.05 mL bovine plasma samples using TRIzol LS. c Results of RT-qPCR quantification of miR-451 in plasma (mean ± SEM) using different RNA volumes for reverse-transcription; highest reaction efficiency was obtained using 2 μL of RNA in a 10 μL cDNA synthesis reaction
    Figure Legend Snippet: Optimisation of methodology for profiling miRNAs in bovine plasma. a RT-qPCR data plots (with mean ± SEM) showing abundance of the exogenous miRNA, cel-miR-39-3p, in plasma samples after RNA extraction using 3 different kits as indicated. b RNA yield from 1.05 mL bovine plasma samples using TRIzol LS. c Results of RT-qPCR quantification of miR-451 in plasma (mean ± SEM) using different RNA volumes for reverse-transcription; highest reaction efficiency was obtained using 2 μL of RNA in a 10 μL cDNA synthesis reaction

    Techniques Used: Quantitative RT-PCR, RNA Extraction

    6) Product Images from "Nuclear Outsourcing of RNA Interference Components to Human Mitochondria"

    Article Title: Nuclear Outsourcing of RNA Interference Components to Human Mitochondria

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020746

    Co-immunoprecipitation of AGO2 and mitochondrial transcripts. Either AGO2, or SLUG, which serves as a negative control were co-immunoprecipitated with associated mRNAs in HeLa protein extracts. Coimmunoprecipitated RNA was extracted with Trizol and subjected to RT-PCR amplification with the indicated primers: cytochrome c oxidase III ( COX3 ), cytochrome b ( cyt b ) and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ). Results are indicative of three independent experiments.
    Figure Legend Snippet: Co-immunoprecipitation of AGO2 and mitochondrial transcripts. Either AGO2, or SLUG, which serves as a negative control were co-immunoprecipitated with associated mRNAs in HeLa protein extracts. Coimmunoprecipitated RNA was extracted with Trizol and subjected to RT-PCR amplification with the indicated primers: cytochrome c oxidase III ( COX3 ), cytochrome b ( cyt b ) and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ). Results are indicative of three independent experiments.

    Techniques Used: Immunoprecipitation, Negative Control, Reverse Transcription Polymerase Chain Reaction, Amplification

    7) Product Images from "MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA"

    Article Title: MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA

    Journal: Journal of Extracellular Vesicles

    doi: 10.3402/jev.v5.29828

    (a) Western blot analysis of CTB-, AV- and ST-bound MSC EVs. MSC CM was incubated with CTB, AV or ST followed by incubation with Dynabeads conjugated with Streptavidin. The beads were immobilised with a magnet, washed, denatured and resolved onto polyacrylamide gels before electroblotting onto a nitrocellulose membrane. The membrane was probed with a primary antibody followed by horseradish peroxidase-coupled secondary antibodies against the primary antibody. The blot was then incubated with a chemiluminescent HRP substrate to detect bound primary antibody. (b) 10 µg MSC EV was extracted sequentially with biotinylated CTB and then biotinylated AV or vice versa. After each extraction, the ligand-bound vesicles were removed with Dynabeads ® MyOne Streptavidin T1 and assayed for CD81 by ELISA. The relative level of CD81 in CTB-vesicles before and after extraction with AV, and that in AV-vesicles before and after extraction with CTB were normalized to that in AV-vesicles before CTB extraction. (c) RNA analysis of CTB-, AV- and ST-EVs. CTB-, AV- or ST-binding EVs were isolated as described above and extracted for RNA using Trizol. The pellet in each of extracts was re-suspended in 50 µL of RNase-free water. 10 µL of each RNA solution was resolved on a 15% Novex Tris-borate-EDTA(TBE)-urea gel before staining with ethidium bromide.
    Figure Legend Snippet: (a) Western blot analysis of CTB-, AV- and ST-bound MSC EVs. MSC CM was incubated with CTB, AV or ST followed by incubation with Dynabeads conjugated with Streptavidin. The beads were immobilised with a magnet, washed, denatured and resolved onto polyacrylamide gels before electroblotting onto a nitrocellulose membrane. The membrane was probed with a primary antibody followed by horseradish peroxidase-coupled secondary antibodies against the primary antibody. The blot was then incubated with a chemiluminescent HRP substrate to detect bound primary antibody. (b) 10 µg MSC EV was extracted sequentially with biotinylated CTB and then biotinylated AV or vice versa. After each extraction, the ligand-bound vesicles were removed with Dynabeads ® MyOne Streptavidin T1 and assayed for CD81 by ELISA. The relative level of CD81 in CTB-vesicles before and after extraction with AV, and that in AV-vesicles before and after extraction with CTB were normalized to that in AV-vesicles before CTB extraction. (c) RNA analysis of CTB-, AV- and ST-EVs. CTB-, AV- or ST-binding EVs were isolated as described above and extracted for RNA using Trizol. The pellet in each of extracts was re-suspended in 50 µL of RNase-free water. 10 µL of each RNA solution was resolved on a 15% Novex Tris-borate-EDTA(TBE)-urea gel before staining with ethidium bromide.

    Techniques Used: Western Blot, CtB Assay, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Isolation, Staining

    8) Product Images from "Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses"

    Article Title: Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.01.002

    Ex-miRNA Are Stable and Protected from RNases in BALF (A) qPCR of synthetic miRNAs (“calibrators” Cal1 and Cal2) spiked into Trizol or BALF with or without an RNase inhibitor (RNAsin) (n = 3 from 1–2 independent experiments, 1-way ANOVA with Bonferroni’s multiple comparison test). (B) qPCR of analysis of miRNAs from BALF and RNA pellets treated with RNaseA (n = 3 from 3 independent experiments, 2-way ANOVA with Bonferroni’s multiple comparison test). Error bars are mean + SD; *p
    Figure Legend Snippet: Ex-miRNA Are Stable and Protected from RNases in BALF (A) qPCR of synthetic miRNAs (“calibrators” Cal1 and Cal2) spiked into Trizol or BALF with or without an RNase inhibitor (RNAsin) (n = 3 from 1–2 independent experiments, 1-way ANOVA with Bonferroni’s multiple comparison test). (B) qPCR of analysis of miRNAs from BALF and RNA pellets treated with RNaseA (n = 3 from 3 independent experiments, 2-way ANOVA with Bonferroni’s multiple comparison test). Error bars are mean + SD; *p

    Techniques Used: Real-time Polymerase Chain Reaction

    9) Product Images from "Effects of amino acid substitutions in the VP2 B-C loop on antigenicity and pathogenicity of serotype Asia1 foot-and-mouth disease virus"

    Article Title: Effects of amino acid substitutions in the VP2 B-C loop on antigenicity and pathogenicity of serotype Asia1 foot-and-mouth disease virus

    Journal: Virology Journal

    doi: 10.1186/1743-422X-9-191

    Growth kinetics of rD72G and rD72N mutant viruses. BHK-21 cells were infected and harvested at different times post infection. ( A ) Single-step growth curves obtained by titration of virus present at each time. Samples were taken in triplicates and standard deviations are shown in the graphic. ( B ) RNA production curves were obtained by real time RT-PCR assay from Trizol resuspended monolayers. The mean viral load generated by real time RT-PCR is expressed as log 10 vRNA copies/ml of virus transport medium. Samples were taken in triplicates and standard deviations are shown in the graphic. (◆) FMDV Asia1/YS/CHA/05,(■) rD72G and (▴) rD72N.
    Figure Legend Snippet: Growth kinetics of rD72G and rD72N mutant viruses. BHK-21 cells were infected and harvested at different times post infection. ( A ) Single-step growth curves obtained by titration of virus present at each time. Samples were taken in triplicates and standard deviations are shown in the graphic. ( B ) RNA production curves were obtained by real time RT-PCR assay from Trizol resuspended monolayers. The mean viral load generated by real time RT-PCR is expressed as log 10 vRNA copies/ml of virus transport medium. Samples were taken in triplicates and standard deviations are shown in the graphic. (◆) FMDV Asia1/YS/CHA/05,(■) rD72G and (▴) rD72N.

    Techniques Used: Mutagenesis, Infection, Titration, Quantitative RT-PCR, Generated

    10) Product Images from "Rapid Detection of Human Pathogenic Orthobunyaviruses"

    Article Title: Rapid Detection of Human Pathogenic Orthobunyaviruses

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.41.7.3299-3305.2003

    Detection of OROV in patient sera. (A) OROV RNA standard range from 10 7 to 10 1 molecules (as established on the SmartCycler). (B) Results are given as the number of copies of viral RNA detected per milliliter of serum obtained from each of 28 patients sampled on days 1 to 5 after onset of disease. Two patient samples drawn on days 2 and 3 after onset of disease tested negative. RNA was Trizol extracted from 125 μl of serum. OROV infection had been previously confirmed by virus isolation.
    Figure Legend Snippet: Detection of OROV in patient sera. (A) OROV RNA standard range from 10 7 to 10 1 molecules (as established on the SmartCycler). (B) Results are given as the number of copies of viral RNA detected per milliliter of serum obtained from each of 28 patients sampled on days 1 to 5 after onset of disease. Two patient samples drawn on days 2 and 3 after onset of disease tested negative. RNA was Trizol extracted from 125 μl of serum. OROV infection had been previously confirmed by virus isolation.

    Techniques Used: Infection, Virus Isolation Assay

    11) Product Images from "Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses"

    Article Title: Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.01.002

    Ex-miRNA Are Stable and Protected from RNases in BALF (A) qPCR of synthetic miRNAs (“calibrators” Cal1 and Cal2) spiked into Trizol or BALF with or without an RNase inhibitor (RNAsin) (n = 3 from 1–2 independent experiments, 1-way ANOVA with Bonferroni’s multiple comparison test). (B) qPCR of analysis of miRNAs from BALF and RNA pellets treated with RNaseA (n = 3 from 3 independent experiments, 2-way ANOVA with Bonferroni’s multiple comparison test). Error bars are mean + SD; *p
    Figure Legend Snippet: Ex-miRNA Are Stable and Protected from RNases in BALF (A) qPCR of synthetic miRNAs (“calibrators” Cal1 and Cal2) spiked into Trizol or BALF with or without an RNase inhibitor (RNAsin) (n = 3 from 1–2 independent experiments, 1-way ANOVA with Bonferroni’s multiple comparison test). (B) qPCR of analysis of miRNAs from BALF and RNA pellets treated with RNaseA (n = 3 from 3 independent experiments, 2-way ANOVA with Bonferroni’s multiple comparison test). Error bars are mean + SD; *p

    Techniques Used: Real-time Polymerase Chain Reaction

    12) Product Images from "Reactivation Kinetics of HIV-1 and Susceptibility of Reactivated Latently Infected CD4+ T Cells to HIV-1-Specific CD8+ T Cells"

    Article Title: Reactivation Kinetics of HIV-1 and Susceptibility of Reactivated Latently Infected CD4+ T Cells to HIV-1-Specific CD8+ T Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.01454-15

    HIV-1 mRNA is upregulated in primary resting CD4 + T cells 1 h poststimulation with PMA and ionomycin. Primary, resting CD4 + T cells were treated with PMA and ionomycin in the presence of raltegravir and efavirenz for 6 h with supernatant and cellular samples taken before and 1, 3, and 6 h after initiation of stimulation. Intracellular and supernatant RNA was isolated with TRIzol and converted to cDNA, of which the HIV-1 mRNA was then quantified via qPCR. Intracellular HIV-1 mRNA is upregulated in vitro after 1 h of PMA and ionomycin treatment. Solid symbols represent detectable RNA, and open symbols represent undetectable RNA levels. The dashed line represents the limit of detection (3.33 to 16.89 copies per million cells).
    Figure Legend Snippet: HIV-1 mRNA is upregulated in primary resting CD4 + T cells 1 h poststimulation with PMA and ionomycin. Primary, resting CD4 + T cells were treated with PMA and ionomycin in the presence of raltegravir and efavirenz for 6 h with supernatant and cellular samples taken before and 1, 3, and 6 h after initiation of stimulation. Intracellular and supernatant RNA was isolated with TRIzol and converted to cDNA, of which the HIV-1 mRNA was then quantified via qPCR. Intracellular HIV-1 mRNA is upregulated in vitro after 1 h of PMA and ionomycin treatment. Solid symbols represent detectable RNA, and open symbols represent undetectable RNA levels. The dashed line represents the limit of detection (3.33 to 16.89 copies per million cells).

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, In Vitro

    13) Product Images from "Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum"

    Article Title: Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2017.3080

    Flowchart of the study design. Exosomes from cell culture medium (CCM) were isolated using ultracentrifugation (UC), ExoQuick-TC and Total Exosome Isolation reagent for CCM (TEI-A). Exosomal RNA (ExoRNA) was subsequently isolated using TRIzol-LS, SeraMir, HiPure Liquid RNA/miRNA kit (HLR) and Total Exosome RNA Isolation (TER). Exosomes from serum were isolated using UC, ExoQuick and TEI-B for serum (TEI-B). ExoRNA was subsequently isolated using TRIzol-LS, SeraMir, HLR, miRNeasy, exoRNeasy and TER. Route_1 to Route_5 (for CCM) and Route_a to Route_f (for serum) represent different combinations of isolation methods for exosomes and exoRNA. Red color highlights the recommended methods, which are discussed in the text.
    Figure Legend Snippet: Flowchart of the study design. Exosomes from cell culture medium (CCM) were isolated using ultracentrifugation (UC), ExoQuick-TC and Total Exosome Isolation reagent for CCM (TEI-A). Exosomal RNA (ExoRNA) was subsequently isolated using TRIzol-LS, SeraMir, HiPure Liquid RNA/miRNA kit (HLR) and Total Exosome RNA Isolation (TER). Exosomes from serum were isolated using UC, ExoQuick and TEI-B for serum (TEI-B). ExoRNA was subsequently isolated using TRIzol-LS, SeraMir, HLR, miRNeasy, exoRNeasy and TER. Route_1 to Route_5 (for CCM) and Route_a to Route_f (for serum) represent different combinations of isolation methods for exosomes and exoRNA. Red color highlights the recommended methods, which are discussed in the text.

    Techniques Used: Cell Culture, Isolation

    14) Product Images from "Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients"

    Article Title: Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients

    Journal: JCI Insight

    doi: 10.1172/jci.insight.89631

    Candidate miRNA levels are elevated in EVs of cHL patients compared with healthy controls. RT-PCR analysis of miR127-3p ( A ), miR155-5p ( B ), miR21-5p ( C ), let7a-5p ( D ), miR24-3p ( E ), and miR10b-5p ( F ) in plasma extracellular vesicles (EVs) of healthy individuals ( n = 9) and cHL patients ( n = 20) after size-exclusion chromatography (SEC) and total RNA isolation using TRIzol. For each individual sample, the mean Ct value of SEC fractions 9 and 10 was used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. * P
    Figure Legend Snippet: Candidate miRNA levels are elevated in EVs of cHL patients compared with healthy controls. RT-PCR analysis of miR127-3p ( A ), miR155-5p ( B ), miR21-5p ( C ), let7a-5p ( D ), miR24-3p ( E ), and miR10b-5p ( F ) in plasma extracellular vesicles (EVs) of healthy individuals ( n = 9) and cHL patients ( n = 20) after size-exclusion chromatography (SEC) and total RNA isolation using TRIzol. For each individual sample, the mean Ct value of SEC fractions 9 and 10 was used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. * P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Size-exclusion Chromatography, Isolation

    EV outperforms total plasma for monitoring treatment response and corresponds with TARC. ( A ) RT-PCR analysis of miR127-3p in total plasma of cHL patients ( n = 7) before and after treatment, after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in plasma extracellular vesicles (EVs) of the same cHL patients ( n = 7) as in A , after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ( C and D ) As in A and B , but for miR155-5p. ( E and F ) RT-PCR analysis of miR21-5p, miR155-5p, and miR127-3p in total plasma ( E ) and in plasma EVs ( F ) of an individual cHL patient with primary tumor before and after first-line treatment (gray symbols) and a cHL patient with relapsed disease before and after second-line treatment (black symbols). ( G – J ) RT-PCR analysis of miR127-3p ( G ), miR155-5p ( H ), miR21-5p ( I ), and let7a-5p ( J ) in plasma EVs of cHL patients before and after treatment ( n = 7). Each data point is the mean Ct value of the 2 consecutive SEC fractions 9 and 10. ( K ) Serum TARC levels in the same cHL patients as in G–J before and after treatment, as measured by ELISA. Data are shown as paired before and after therapy samples ( E–K ).
    Figure Legend Snippet: EV outperforms total plasma for monitoring treatment response and corresponds with TARC. ( A ) RT-PCR analysis of miR127-3p in total plasma of cHL patients ( n = 7) before and after treatment, after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in plasma extracellular vesicles (EVs) of the same cHL patients ( n = 7) as in A , after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ( C and D ) As in A and B , but for miR155-5p. ( E and F ) RT-PCR analysis of miR21-5p, miR155-5p, and miR127-3p in total plasma ( E ) and in plasma EVs ( F ) of an individual cHL patient with primary tumor before and after first-line treatment (gray symbols) and a cHL patient with relapsed disease before and after second-line treatment (black symbols). ( G – J ) RT-PCR analysis of miR127-3p ( G ), miR155-5p ( H ), miR21-5p ( I ), and let7a-5p ( J ) in plasma EVs of cHL patients before and after treatment ( n = 7). Each data point is the mean Ct value of the 2 consecutive SEC fractions 9 and 10. ( K ) Serum TARC levels in the same cHL patients as in G–J before and after treatment, as measured by ELISA. Data are shown as paired before and after therapy samples ( E–K ).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay

    Small RNA distribution and recovery in EV fractions 9 and 10. ( A and B ) RNA distribution of miR142-3p, let7a-5p, and vtRNA1-1 ( A ) and miR92a-3p, miR21-5p, and miR451-5p ( B ) in 26 fractions upon size-exclusion chromatography (SEC) of 1.5 ml healthy donor plasma. Total RNA was isolated with TRIzol followed by RT-PCR. Data are depicted as raw Ct values; error bars represent SEM from PCR duplicates. ( C ) Fold enrichment of vtRNA1-1, let7a-5p, and miR142-3p in plasma extracellular vesicles (EVs) (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of 2 donors; dots indicate individual samples. ( D ) Fold enrichment of miR92a-3p, miR21-5p, and miR451-5p in protein/HDL (fractions 20 and 21) compared with plasma EVs (fractions 9 and 10). Data are shown as the mean of 2 donors; dots indicate individual samples. ( E ) Fold enrichment of vtRNA1-1 in tumor EV (tEV; fractions 9 and 10) compared with protein/HDL (fractions 20 and 21) after SEC of 1.5 ml B cell culture supernatant. ( F ) SEC of 1.5 ml healthy donor plasma after spike in with 50 μl tumor cell line–derived exosomes. Shown is the fold increase of EBV-miR BHRF1-3 and BART2-5p in EV (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of the 2 consecutive SEC fractions; dots represent individual fractions ( E and F ).
    Figure Legend Snippet: Small RNA distribution and recovery in EV fractions 9 and 10. ( A and B ) RNA distribution of miR142-3p, let7a-5p, and vtRNA1-1 ( A ) and miR92a-3p, miR21-5p, and miR451-5p ( B ) in 26 fractions upon size-exclusion chromatography (SEC) of 1.5 ml healthy donor plasma. Total RNA was isolated with TRIzol followed by RT-PCR. Data are depicted as raw Ct values; error bars represent SEM from PCR duplicates. ( C ) Fold enrichment of vtRNA1-1, let7a-5p, and miR142-3p in plasma extracellular vesicles (EVs) (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of 2 donors; dots indicate individual samples. ( D ) Fold enrichment of miR92a-3p, miR21-5p, and miR451-5p in protein/HDL (fractions 20 and 21) compared with plasma EVs (fractions 9 and 10). Data are shown as the mean of 2 donors; dots indicate individual samples. ( E ) Fold enrichment of vtRNA1-1 in tumor EV (tEV; fractions 9 and 10) compared with protein/HDL (fractions 20 and 21) after SEC of 1.5 ml B cell culture supernatant. ( F ) SEC of 1.5 ml healthy donor plasma after spike in with 50 μl tumor cell line–derived exosomes. Shown is the fold increase of EBV-miR BHRF1-3 and BART2-5p in EV (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of the 2 consecutive SEC fractions; dots represent individual fractions ( E and F ).

    Techniques Used: Size-exclusion Chromatography, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Cell Culture, Derivative Assay

    miR127-3p EV outperforms total plasma in distinguishing cHL patients from controls. ( A ) RT-PCR analysis of miR127-3p in total plasma of healthy controls ( n = 7) and cHL patients ( n = 8) after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in extracellular vesicle (EV) fractions of the same healthy individuals and cHL patients as in A after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. ( A and B ) Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ** P
    Figure Legend Snippet: miR127-3p EV outperforms total plasma in distinguishing cHL patients from controls. ( A ) RT-PCR analysis of miR127-3p in total plasma of healthy controls ( n = 7) and cHL patients ( n = 8) after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in extracellular vesicle (EV) fractions of the same healthy individuals and cHL patients as in A after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. ( A and B ) Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ** P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Size-exclusion Chromatography

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses
    Article Snippet: .. qPCR Total RNA was isolated from fluids using Trizol LS (Invitrogen) and cells or vesicle pellets using Trizol (Invitrogen). .. Small RNA reverse transcription was performed using the miR-X First Strand Synthesis kit (Clonetech).

    Article Title: GPER-independent inhibition of adrenocortical cancer growth by G-1 involves ROS/Egr-1/BAX pathway
    Article Snippet: .. RNA extraction, reverse transcription and real time PCR TRizol RNA isolation system (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from H295R. .. Each RNA sample was treated with DNase I (Invitrogen), and purity and integrity of the RNA were confirmed spectroscopically and by gel electrophoresis before use.

    Isolation:

    Article Title: Increased Hematopoietic Extracellular RNAs and Vesicles in the Lung during Allergic Airway Responses
    Article Snippet: .. qPCR Total RNA was isolated from fluids using Trizol LS (Invitrogen) and cells or vesicle pellets using Trizol (Invitrogen). .. Small RNA reverse transcription was performed using the miR-X First Strand Synthesis kit (Clonetech).

    Article Title: Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods
    Article Snippet: .. For total RNA extraction, 5 different methods were tested: miRNeasy Mini kit (#217004 Qiagen), miRCURY RNA Isolation kit (#300110 Exiqon, Vedbaek, DK), Allprep DNA/RNA/miRNA Universal kit (#80224 Qiagen), TRIzol LS (#10296010 Life Technologies) and TRIzol (#15596026 Life Technologies). .. Each RNA extraction was performed from 100 µL of FHHNC patient’s uEVs.

    Article Title: GPER-independent inhibition of adrenocortical cancer growth by G-1 involves ROS/Egr-1/BAX pathway
    Article Snippet: .. RNA extraction, reverse transcription and real time PCR TRizol RNA isolation system (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from H295R. .. Each RNA sample was treated with DNase I (Invitrogen), and purity and integrity of the RNA were confirmed spectroscopically and by gel electrophoresis before use.

    Article Title: MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA
    Article Snippet: .. The isolated EVs were resuspended in 100 µL of PBS and extracted for RNA using 3 volumes of Trizol LS (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol. ..

    Article Title: Evaluation of Pre-Analytical Variables in the Quantification of Dengue Virus by Real-Time Polymerase Chain Reaction
    Article Snippet: .. Viral RNA was extracted with TRIzol LS (Invitrogen, CA) in the presence or absence of linear acrylamide (20 μg/ml, Ambion, Tx) as a co-precipitant, High Pure Viral RNA Isolation Kit (Roche Applied Sciences, Germany) or QIAamp Viral RNA Kit (Qiagen, Germany). .. Briefly, physiologically relevant titrated concentrations of DENV virions (1 × 104 plaque forming units [PFUs]/ml) or genomic RNA (105 to 107 copies/ml) were added to commercially available normal human sera or PBS that were premixed with the lysis/binding buffer to prevent viral RNA degradation before extraction.

    RNA Extraction:

    Article Title: Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods
    Article Snippet: .. For total RNA extraction, 5 different methods were tested: miRNeasy Mini kit (#217004 Qiagen), miRCURY RNA Isolation kit (#300110 Exiqon, Vedbaek, DK), Allprep DNA/RNA/miRNA Universal kit (#80224 Qiagen), TRIzol LS (#10296010 Life Technologies) and TRIzol (#15596026 Life Technologies). .. Each RNA extraction was performed from 100 µL of FHHNC patient’s uEVs.

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    Article Snippet: .. RNA extraction, reverse transcription and real time PCR TRizol RNA isolation system (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from H295R. .. Each RNA sample was treated with DNase I (Invitrogen), and purity and integrity of the RNA were confirmed spectroscopically and by gel electrophoresis before use.

    RNA Sequencing Assay:

    Article Title: Alterations in the host transcriptome in vitro following Rift Valley fever virus infection
    Article Snippet: .. RNA Sequencing HSAEC samples infected with RVFV MP12 or ZH548 were collected at 3, 9, and 18 hours post infection in TRIzol LS (Ambion, 10296010). .. RNA was extracted per the manufacturer’s instructions and quantified using the LabChipGX (Perkin Elmer).

    Infection:

    Article Title: Alterations in the host transcriptome in vitro following Rift Valley fever virus infection
    Article Snippet: .. RNA Sequencing HSAEC samples infected with RVFV MP12 or ZH548 were collected at 3, 9, and 18 hours post infection in TRIzol LS (Ambion, 10296010). .. RNA was extracted per the manufacturer’s instructions and quantified using the LabChipGX (Perkin Elmer).

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    Thermo Fisher trizol ls
    Candidate miRNA levels are elevated in EVs of cHL patients compared with healthy controls. RT-PCR analysis of miR127-3p ( A ), miR155-5p ( B ), miR21-5p ( C ), let7a-5p ( D ), miR24-3p ( E ), and miR10b-5p ( F ) in plasma extracellular vesicles (EVs) of healthy individuals ( n = 9) and cHL patients ( n = 20) after size-exclusion chromatography (SEC) and total <t>RNA</t> isolation using <t>TRIzol.</t> For each individual sample, the mean Ct value of SEC fractions 9 and 10 was used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. * P
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    Candidate miRNA levels are elevated in EVs of cHL patients compared with healthy controls. RT-PCR analysis of miR127-3p ( A ), miR155-5p ( B ), miR21-5p ( C ), let7a-5p ( D ), miR24-3p ( E ), and miR10b-5p ( F ) in plasma extracellular vesicles (EVs) of healthy individuals ( n = 9) and cHL patients ( n = 20) after size-exclusion chromatography (SEC) and total RNA isolation using TRIzol. For each individual sample, the mean Ct value of SEC fractions 9 and 10 was used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. * P

    Journal: JCI Insight

    Article Title: Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients

    doi: 10.1172/jci.insight.89631

    Figure Lengend Snippet: Candidate miRNA levels are elevated in EVs of cHL patients compared with healthy controls. RT-PCR analysis of miR127-3p ( A ), miR155-5p ( B ), miR21-5p ( C ), let7a-5p ( D ), miR24-3p ( E ), and miR10b-5p ( F ) in plasma extracellular vesicles (EVs) of healthy individuals ( n = 9) and cHL patients ( n = 20) after size-exclusion chromatography (SEC) and total RNA isolation using TRIzol. For each individual sample, the mean Ct value of SEC fractions 9 and 10 was used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. * P

    Article Snippet: For RNA isolation of total plasma or sera, 0.75 ml TRIzol-LS (Thermo Fisher Scientific) was added to 0.25 ml plasma and further processed as described above.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Size-exclusion Chromatography, Isolation

    EV outperforms total plasma for monitoring treatment response and corresponds with TARC. ( A ) RT-PCR analysis of miR127-3p in total plasma of cHL patients ( n = 7) before and after treatment, after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in plasma extracellular vesicles (EVs) of the same cHL patients ( n = 7) as in A , after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ( C and D ) As in A and B , but for miR155-5p. ( E and F ) RT-PCR analysis of miR21-5p, miR155-5p, and miR127-3p in total plasma ( E ) and in plasma EVs ( F ) of an individual cHL patient with primary tumor before and after first-line treatment (gray symbols) and a cHL patient with relapsed disease before and after second-line treatment (black symbols). ( G – J ) RT-PCR analysis of miR127-3p ( G ), miR155-5p ( H ), miR21-5p ( I ), and let7a-5p ( J ) in plasma EVs of cHL patients before and after treatment ( n = 7). Each data point is the mean Ct value of the 2 consecutive SEC fractions 9 and 10. ( K ) Serum TARC levels in the same cHL patients as in G–J before and after treatment, as measured by ELISA. Data are shown as paired before and after therapy samples ( E–K ).

    Journal: JCI Insight

    Article Title: Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients

    doi: 10.1172/jci.insight.89631

    Figure Lengend Snippet: EV outperforms total plasma for monitoring treatment response and corresponds with TARC. ( A ) RT-PCR analysis of miR127-3p in total plasma of cHL patients ( n = 7) before and after treatment, after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in plasma extracellular vesicles (EVs) of the same cHL patients ( n = 7) as in A , after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ( C and D ) As in A and B , but for miR155-5p. ( E and F ) RT-PCR analysis of miR21-5p, miR155-5p, and miR127-3p in total plasma ( E ) and in plasma EVs ( F ) of an individual cHL patient with primary tumor before and after first-line treatment (gray symbols) and a cHL patient with relapsed disease before and after second-line treatment (black symbols). ( G – J ) RT-PCR analysis of miR127-3p ( G ), miR155-5p ( H ), miR21-5p ( I ), and let7a-5p ( J ) in plasma EVs of cHL patients before and after treatment ( n = 7). Each data point is the mean Ct value of the 2 consecutive SEC fractions 9 and 10. ( K ) Serum TARC levels in the same cHL patients as in G–J before and after treatment, as measured by ELISA. Data are shown as paired before and after therapy samples ( E–K ).

    Article Snippet: For RNA isolation of total plasma or sera, 0.75 ml TRIzol-LS (Thermo Fisher Scientific) was added to 0.25 ml plasma and further processed as described above.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay

    Small RNA distribution and recovery in EV fractions 9 and 10. ( A and B ) RNA distribution of miR142-3p, let7a-5p, and vtRNA1-1 ( A ) and miR92a-3p, miR21-5p, and miR451-5p ( B ) in 26 fractions upon size-exclusion chromatography (SEC) of 1.5 ml healthy donor plasma. Total RNA was isolated with TRIzol followed by RT-PCR. Data are depicted as raw Ct values; error bars represent SEM from PCR duplicates. ( C ) Fold enrichment of vtRNA1-1, let7a-5p, and miR142-3p in plasma extracellular vesicles (EVs) (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of 2 donors; dots indicate individual samples. ( D ) Fold enrichment of miR92a-3p, miR21-5p, and miR451-5p in protein/HDL (fractions 20 and 21) compared with plasma EVs (fractions 9 and 10). Data are shown as the mean of 2 donors; dots indicate individual samples. ( E ) Fold enrichment of vtRNA1-1 in tumor EV (tEV; fractions 9 and 10) compared with protein/HDL (fractions 20 and 21) after SEC of 1.5 ml B cell culture supernatant. ( F ) SEC of 1.5 ml healthy donor plasma after spike in with 50 μl tumor cell line–derived exosomes. Shown is the fold increase of EBV-miR BHRF1-3 and BART2-5p in EV (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of the 2 consecutive SEC fractions; dots represent individual fractions ( E and F ).

    Journal: JCI Insight

    Article Title: Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients

    doi: 10.1172/jci.insight.89631

    Figure Lengend Snippet: Small RNA distribution and recovery in EV fractions 9 and 10. ( A and B ) RNA distribution of miR142-3p, let7a-5p, and vtRNA1-1 ( A ) and miR92a-3p, miR21-5p, and miR451-5p ( B ) in 26 fractions upon size-exclusion chromatography (SEC) of 1.5 ml healthy donor plasma. Total RNA was isolated with TRIzol followed by RT-PCR. Data are depicted as raw Ct values; error bars represent SEM from PCR duplicates. ( C ) Fold enrichment of vtRNA1-1, let7a-5p, and miR142-3p in plasma extracellular vesicles (EVs) (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of 2 donors; dots indicate individual samples. ( D ) Fold enrichment of miR92a-3p, miR21-5p, and miR451-5p in protein/HDL (fractions 20 and 21) compared with plasma EVs (fractions 9 and 10). Data are shown as the mean of 2 donors; dots indicate individual samples. ( E ) Fold enrichment of vtRNA1-1 in tumor EV (tEV; fractions 9 and 10) compared with protein/HDL (fractions 20 and 21) after SEC of 1.5 ml B cell culture supernatant. ( F ) SEC of 1.5 ml healthy donor plasma after spike in with 50 μl tumor cell line–derived exosomes. Shown is the fold increase of EBV-miR BHRF1-3 and BART2-5p in EV (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of the 2 consecutive SEC fractions; dots represent individual fractions ( E and F ).

    Article Snippet: For RNA isolation of total plasma or sera, 0.75 ml TRIzol-LS (Thermo Fisher Scientific) was added to 0.25 ml plasma and further processed as described above.

    Techniques: Size-exclusion Chromatography, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Cell Culture, Derivative Assay

    miR127-3p EV outperforms total plasma in distinguishing cHL patients from controls. ( A ) RT-PCR analysis of miR127-3p in total plasma of healthy controls ( n = 7) and cHL patients ( n = 8) after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in extracellular vesicle (EV) fractions of the same healthy individuals and cHL patients as in A after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. ( A and B ) Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ** P

    Journal: JCI Insight

    Article Title: Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients

    doi: 10.1172/jci.insight.89631

    Figure Lengend Snippet: miR127-3p EV outperforms total plasma in distinguishing cHL patients from controls. ( A ) RT-PCR analysis of miR127-3p in total plasma of healthy controls ( n = 7) and cHL patients ( n = 8) after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in extracellular vesicle (EV) fractions of the same healthy individuals and cHL patients as in A after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. ( A and B ) Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ** P

    Article Snippet: For RNA isolation of total plasma or sera, 0.75 ml TRIzol-LS (Thermo Fisher Scientific) was added to 0.25 ml plasma and further processed as described above.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Size-exclusion Chromatography

    (a) Western blot analysis of CTB-, AV- and ST-bound MSC EVs. MSC CM was incubated with CTB, AV or ST followed by incubation with Dynabeads conjugated with Streptavidin. The beads were immobilised with a magnet, washed, denatured and resolved onto polyacrylamide gels before electroblotting onto a nitrocellulose membrane. The membrane was probed with a primary antibody followed by horseradish peroxidase-coupled secondary antibodies against the primary antibody. The blot was then incubated with a chemiluminescent HRP substrate to detect bound primary antibody. (b) 10 µg MSC EV was extracted sequentially with biotinylated CTB and then biotinylated AV or vice versa. After each extraction, the ligand-bound vesicles were removed with Dynabeads ® MyOne Streptavidin T1 and assayed for CD81 by ELISA. The relative level of CD81 in CTB-vesicles before and after extraction with AV, and that in AV-vesicles before and after extraction with CTB were normalized to that in AV-vesicles before CTB extraction. (c) RNA analysis of CTB-, AV- and ST-EVs. CTB-, AV- or ST-binding EVs were isolated as described above and extracted for RNA using Trizol. The pellet in each of extracts was re-suspended in 50 µL of RNase-free water. 10 µL of each RNA solution was resolved on a 15% Novex Tris-borate-EDTA(TBE)-urea gel before staining with ethidium bromide.

    Journal: Journal of Extracellular Vesicles

    Article Title: MSC secretes at least 3 EV types each with a unique permutation of membrane lipid, protein and RNA

    doi: 10.3402/jev.v5.29828

    Figure Lengend Snippet: (a) Western blot analysis of CTB-, AV- and ST-bound MSC EVs. MSC CM was incubated with CTB, AV or ST followed by incubation with Dynabeads conjugated with Streptavidin. The beads were immobilised with a magnet, washed, denatured and resolved onto polyacrylamide gels before electroblotting onto a nitrocellulose membrane. The membrane was probed with a primary antibody followed by horseradish peroxidase-coupled secondary antibodies against the primary antibody. The blot was then incubated with a chemiluminescent HRP substrate to detect bound primary antibody. (b) 10 µg MSC EV was extracted sequentially with biotinylated CTB and then biotinylated AV or vice versa. After each extraction, the ligand-bound vesicles were removed with Dynabeads ® MyOne Streptavidin T1 and assayed for CD81 by ELISA. The relative level of CD81 in CTB-vesicles before and after extraction with AV, and that in AV-vesicles before and after extraction with CTB were normalized to that in AV-vesicles before CTB extraction. (c) RNA analysis of CTB-, AV- and ST-EVs. CTB-, AV- or ST-binding EVs were isolated as described above and extracted for RNA using Trizol. The pellet in each of extracts was re-suspended in 50 µL of RNase-free water. 10 µL of each RNA solution was resolved on a 15% Novex Tris-borate-EDTA(TBE)-urea gel before staining with ethidium bromide.

    Article Snippet: The isolated EVs were resuspended in 100 µL of PBS and extracted for RNA using 3 volumes of Trizol LS (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol.

    Techniques: Western Blot, CtB Assay, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Isolation, Staining

    Characterization of HBV DNA and RNA in sera of CHB patients. (A and B) Analyses of serum viral DNA from CHB patients by Southern blotting. Viral DNA was extracted from serum samples obtained from forty-five chronic hepatitis B patients (20% of input sample used for protein A/G agarose beads pulldown) and subjected to Southern blot analysis. Alternatively, these samples were first incubated with protein A/G agarose beads, and then viral DNA in the pulldown mixtures was analyzed by Southern blotting. Serum samples selected for further examining are marked with arrows, and samples with SS DNA detection are labeled with asterisks. (C) Protein A/G agarose bead pulldown of viral particles. Sera (25 μl each) from CHB patients 37, 38, 14, and 35 (M1, mixture one) or from patients 17, 21, 42, and 44 (M2, mixture two) were pooled and incubated with protein A/G agarose beads. Viral DNA in input sera, protein A/G bead pulldown mixtures (beads), and the remaining supernatants (sup.) were extracted and subjected to Southern blot analysis. (D) Northern blot detection of serum viral RNA from patients 37, 38, 14, 35, 17, 21, 42, and 44. Total RNA were extracted from serum samples by TRIzol reagent and treated with DNase I before Northern blot analysis. (E to G) Southern blot analyses of viral DNA from selected samples. Viral DNA was separated by electrophoresis through TAE or alkaline agarose gels, followed by Southern blot detection with the indicated riboprobes.

    Journal: Journal of Virology

    Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

    doi: 10.1128/JVI.00798-18

    Figure Lengend Snippet: Characterization of HBV DNA and RNA in sera of CHB patients. (A and B) Analyses of serum viral DNA from CHB patients by Southern blotting. Viral DNA was extracted from serum samples obtained from forty-five chronic hepatitis B patients (20% of input sample used for protein A/G agarose beads pulldown) and subjected to Southern blot analysis. Alternatively, these samples were first incubated with protein A/G agarose beads, and then viral DNA in the pulldown mixtures was analyzed by Southern blotting. Serum samples selected for further examining are marked with arrows, and samples with SS DNA detection are labeled with asterisks. (C) Protein A/G agarose bead pulldown of viral particles. Sera (25 μl each) from CHB patients 37, 38, 14, and 35 (M1, mixture one) or from patients 17, 21, 42, and 44 (M2, mixture two) were pooled and incubated with protein A/G agarose beads. Viral DNA in input sera, protein A/G bead pulldown mixtures (beads), and the remaining supernatants (sup.) were extracted and subjected to Southern blot analysis. (D) Northern blot detection of serum viral RNA from patients 37, 38, 14, 35, 17, 21, 42, and 44. Total RNA were extracted from serum samples by TRIzol reagent and treated with DNase I before Northern blot analysis. (E to G) Southern blot analyses of viral DNA from selected samples. Viral DNA was separated by electrophoresis through TAE or alkaline agarose gels, followed by Southern blot detection with the indicated riboprobes.

    Article Snippet: In addition to the SDS-proteinase K method, viral RNA was also extracted with TRIzol LS reagent according to the manufacturer’s instructions (Thermo Fisher Scientific).

    Techniques: Southern Blot, Incubation, Labeling, Northern Blot, Electrophoresis

    ). (B) Identification of 3′ ends of extracellular HBV RNAs. 3′ Ends of extracellular HBV RNAs were identified by the 3′ RACE method using different HBV-specific anchor primers (the same 5′ primers used for generating templates for producing riboprobes used in panel A, lower). Identified 3′ ends were numbered as described above, and numbers in parentheses indicate the amount of clones with the same 3′ ends. The asterisk indicates unknown nucleic acid copurified with intracellular capsid-associated viral RNA by TRIzol reagent. FL, full-length; Cap, 5′ cap of pregenomic RNA; pA, the polyadenylation site; An, poly(A) tail.

    Journal: Journal of Virology

    Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

    doi: 10.1128/JVI.00798-18

    Figure Lengend Snippet: ). (B) Identification of 3′ ends of extracellular HBV RNAs. 3′ Ends of extracellular HBV RNAs were identified by the 3′ RACE method using different HBV-specific anchor primers (the same 5′ primers used for generating templates for producing riboprobes used in panel A, lower). Identified 3′ ends were numbered as described above, and numbers in parentheses indicate the amount of clones with the same 3′ ends. The asterisk indicates unknown nucleic acid copurified with intracellular capsid-associated viral RNA by TRIzol reagent. FL, full-length; Cap, 5′ cap of pregenomic RNA; pA, the polyadenylation site; An, poly(A) tail.

    Article Snippet: In addition to the SDS-proteinase K method, viral RNA was also extracted with TRIzol LS reagent according to the manufacturer’s instructions (Thermo Fisher Scientific).

    Techniques: Clone Assay

    Characterization of HBV DNA and RNA in sera of CHB patients. (A and B) Analyses of serum viral DNA from CHB patients by Southern blotting. Viral DNA was extracted from serum samples obtained from forty-five chronic hepatitis B patients (20% of input sample used for protein A/G agarose beads pulldown) and subjected to Southern blot analysis. Alternatively, these samples were first incubated with protein A/G agarose beads, and then viral DNA in the pulldown mixtures was analyzed by Southern blotting. Serum samples selected for further examining are marked with arrows, and samples with SS DNA detection are labeled with asterisks. (C) Protein A/G agarose bead pulldown of viral particles. Sera (25 μl each) from CHB patients 37, 38, 14, and 35 (M1, mixture one) or from patients 17, 21, 42, and 44 (M2, mixture two) were pooled and incubated with protein A/G agarose beads. Viral DNA in input sera, protein A/G bead pulldown mixtures (beads), and the remaining supernatants (sup.) were extracted and subjected to Southern blot analysis. (D) Northern blot detection of serum viral RNA from patients 37, 38, 14, 35, 17, 21, 42, and 44. Total RNA were extracted from serum samples by TRIzol reagent and treated with DNase I before Northern blot analysis. (E to G) Southern blot analyses of viral DNA from selected samples. Viral DNA was separated by electrophoresis through TAE or alkaline agarose gels, followed by Southern blot detection with the indicated riboprobes.

    Journal: Journal of Virology

    Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

    doi: 10.1128/JVI.00798-18

    Figure Lengend Snippet: Characterization of HBV DNA and RNA in sera of CHB patients. (A and B) Analyses of serum viral DNA from CHB patients by Southern blotting. Viral DNA was extracted from serum samples obtained from forty-five chronic hepatitis B patients (20% of input sample used for protein A/G agarose beads pulldown) and subjected to Southern blot analysis. Alternatively, these samples were first incubated with protein A/G agarose beads, and then viral DNA in the pulldown mixtures was analyzed by Southern blotting. Serum samples selected for further examining are marked with arrows, and samples with SS DNA detection are labeled with asterisks. (C) Protein A/G agarose bead pulldown of viral particles. Sera (25 μl each) from CHB patients 37, 38, 14, and 35 (M1, mixture one) or from patients 17, 21, 42, and 44 (M2, mixture two) were pooled and incubated with protein A/G agarose beads. Viral DNA in input sera, protein A/G bead pulldown mixtures (beads), and the remaining supernatants (sup.) were extracted and subjected to Southern blot analysis. (D) Northern blot detection of serum viral RNA from patients 37, 38, 14, 35, 17, 21, 42, and 44. Total RNA were extracted from serum samples by TRIzol reagent and treated with DNase I before Northern blot analysis. (E to G) Southern blot analyses of viral DNA from selected samples. Viral DNA was separated by electrophoresis through TAE or alkaline agarose gels, followed by Southern blot detection with the indicated riboprobes.

    Article Snippet: In addition to the SDS-proteinase K method, viral RNA was also extracted with TRIzol LS reagent according to the manufacturer’s instructions (Thermo Fisher Scientific).

    Techniques: Southern Blot, Incubation, Labeling, Northern Blot, Electrophoresis

    Mapping and identifying 3′ ends of extracellular HBV RNAs. (A) Northern blot detection of extracellular HBV RNAs with various riboprobes. Viral RNA from cytoplasmic (C) nucleocapsids (lanes 2, 5, 8, 11, 14, and 17) or culture supernatant (S) (lanes 3, 6, 9, 12, 15, and 18) of HepAD38 cells was extracted with TRIzol reagent and treated with DNase I before Northern blot analysis with plus-strand-specific riboprobes spanning the HBV genome as indicated. pgRNA was used as a reference, and map coordinates were numbered according to the sequence of the HBV genome (genotype D, accession number AJ344117.1 ). (B) Identification of 3′ ends of extracellular HBV RNAs. 3′ Ends of extracellular HBV RNAs were identified by the 3′ RACE method using different HBV-specific anchor primers (the same 5′ primers used for generating templates for producing riboprobes used in panel A, lower). Identified 3′ ends were numbered as described above, and numbers in parentheses indicate the amount of clones with the same 3′ ends. The asterisk indicates unknown nucleic acid copurified with intracellular capsid-associated viral RNA by TRIzol reagent. FL, full-length; Cap, 5′ cap of pregenomic RNA; pA, the polyadenylation site; An, poly(A) tail.

    Journal: Journal of Virology

    Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

    doi: 10.1128/JVI.00798-18

    Figure Lengend Snippet: Mapping and identifying 3′ ends of extracellular HBV RNAs. (A) Northern blot detection of extracellular HBV RNAs with various riboprobes. Viral RNA from cytoplasmic (C) nucleocapsids (lanes 2, 5, 8, 11, 14, and 17) or culture supernatant (S) (lanes 3, 6, 9, 12, 15, and 18) of HepAD38 cells was extracted with TRIzol reagent and treated with DNase I before Northern blot analysis with plus-strand-specific riboprobes spanning the HBV genome as indicated. pgRNA was used as a reference, and map coordinates were numbered according to the sequence of the HBV genome (genotype D, accession number AJ344117.1 ). (B) Identification of 3′ ends of extracellular HBV RNAs. 3′ Ends of extracellular HBV RNAs were identified by the 3′ RACE method using different HBV-specific anchor primers (the same 5′ primers used for generating templates for producing riboprobes used in panel A, lower). Identified 3′ ends were numbered as described above, and numbers in parentheses indicate the amount of clones with the same 3′ ends. The asterisk indicates unknown nucleic acid copurified with intracellular capsid-associated viral RNA by TRIzol reagent. FL, full-length; Cap, 5′ cap of pregenomic RNA; pA, the polyadenylation site; An, poly(A) tail.

    Article Snippet: In addition to the SDS-proteinase K method, viral RNA was also extracted with TRIzol LS reagent according to the manufacturer’s instructions (Thermo Fisher Scientific).

    Techniques: Northern Blot, Sequencing, Clone Assay