trizol ls reagent  (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 reagent
    Characterization of HBV DNA and <t>RNA</t> 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 <t>TRIzol</t> 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.
    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 reagent/product/Thermo Fisher
    Average 99 stars, based on 261 article reviews
    Price from $9.99 to $1999.99
    trizol ls reagent - by Bioz Stars, 2020-12
    99/100 stars

    Images

    1) Product Images from "Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients"

    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

    Journal: Journal of Virology

    doi: 10.1128/JVI.00798-18

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Northern Blot, Sequencing, Clone Assay

    2) Product Images from "Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients"

    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

    Journal: Journal of Virology

    doi: 10.1128/JVI.00798-18

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Clone Assay

    3) Product Images from "Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods"

    Article Title: Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-018-1651-z

    RNA quantification. a Bar graph of total RNA quantified from FHHNC uEVs by Bioanalyzer—Picochip using the five different extraction methods. MirCURY kit followed by TRIzol LS were the most efficient methods. b A representative electropherogram shows that uEVs contain small RNA, including microRNAs (10–40 nt). As expected, small amounts of rRNA were detected. c miRNA profiling by RT-qPCR of RNA extracted from FHHNC uEVs. miRNA expression pattern is consistent independently of the RNA extraction method
    Figure Legend Snippet: RNA quantification. a Bar graph of total RNA quantified from FHHNC uEVs by Bioanalyzer—Picochip using the five different extraction methods. MirCURY kit followed by TRIzol LS were the most efficient methods. b A representative electropherogram shows that uEVs contain small RNA, including microRNAs (10–40 nt). As expected, small amounts of rRNA were detected. c miRNA profiling by RT-qPCR of RNA extracted from FHHNC uEVs. miRNA expression pattern is consistent independently of the RNA extraction method

    Techniques Used: Quantitative RT-PCR, Expressing, RNA Extraction

    4) Product Images from "Alterations in the host transcriptome in vitro following Rift Valley fever virus infection"

    Article Title: Alterations in the host transcriptome in vitro following Rift Valley fever virus infection

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14800-3

    Analysis of differentially expressed genes in HSAECs following RVFV infection. ( a ) Venn diagrams depict the upregulated (top panel) and downregulated (bottom panel) genes changed in MP12 only (left), ZH548 only (right), or both (center, gray) at 3, 9, and 18 hours post infection. These genes were changed by 1.5-fold or more and had a p-value ≤ 0.05. ( b ) HSAECs were mock-infected or infected with MP12 or ZH548 at MOI 5 for one hour. Lysates were collected in Trizol LS, RNA was extracted and prepared for RNA sequencing. RNA-sequencing reads were normalized to the total reads, then fold changes were calculated against the uninfected, mock samples at the specified time point. ( c ) RT-qPCR confirmation of some of the top changed transcripts during all time points post infection. HSAECs were mock-infected or infected with MP12 or ZH548 at MOI 5 for one hour. Lysates were collected in Trizol LS, RNA was extracted using the Direct-zol™ RNA MiniPrep, and analyzed for RT-qPCR with TaqMan Gene Expression Assays against IFIT1, IFIT2, IFIT3, and RSAD2. Fold changes were calculated relative to 18 S ribosomal RNA and normalized to mock samples using the ΔΔCt method. Data are expressed as the Mean ± SD (n = 3).
    Figure Legend Snippet: Analysis of differentially expressed genes in HSAECs following RVFV infection. ( a ) Venn diagrams depict the upregulated (top panel) and downregulated (bottom panel) genes changed in MP12 only (left), ZH548 only (right), or both (center, gray) at 3, 9, and 18 hours post infection. These genes were changed by 1.5-fold or more and had a p-value ≤ 0.05. ( b ) HSAECs were mock-infected or infected with MP12 or ZH548 at MOI 5 for one hour. Lysates were collected in Trizol LS, RNA was extracted and prepared for RNA sequencing. RNA-sequencing reads were normalized to the total reads, then fold changes were calculated against the uninfected, mock samples at the specified time point. ( c ) RT-qPCR confirmation of some of the top changed transcripts during all time points post infection. HSAECs were mock-infected or infected with MP12 or ZH548 at MOI 5 for one hour. Lysates were collected in Trizol LS, RNA was extracted using the Direct-zol™ RNA MiniPrep, and analyzed for RT-qPCR with TaqMan Gene Expression Assays against IFIT1, IFIT2, IFIT3, and RSAD2. Fold changes were calculated relative to 18 S ribosomal RNA and normalized to mock samples using the ΔΔCt method. Data are expressed as the Mean ± SD (n = 3).

    Techniques Used: Infection, RNA Sequencing Assay, Quantitative RT-PCR, Expressing

    5) 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

    6) Product Images from "A potential diagnostic marker for ovarian cancer: Involvement of the histone acetyltransferase, human males absent on the first"

    Article Title: A potential diagnostic marker for ovarian cancer: Involvement of the histone acetyltransferase, human males absent on the first

    Journal: Oncology Letters

    doi: 10.3892/ol.2013.1380

    A reduction in hMOF mRNA levels is observed in human ovarian cancer. (A) PCR analysis of 47 clinical ovarian cancer tissues. Total RNA was isolated from the tissues using TRIzol. The PCR assay was performed to detect the mRNA expression levels of hMOF, CA9, VEGF, HIF1α and hSTC1 in clinical ovarian cancer and normal ovarian tissues. The PCR products were then separated by electrophoresis on a 2% agarose gel. The DNA fragments were visualized and photographed under ultraviolet light with ethidium bromide. The mRNA levels from 37 ovarian cancer tissues were compared with corresponding contralateral ovarian normal tissues. However, 10 clinical ovarian cancer tissues were missing contralateral ovarian normal tissues and were compared with non-corresponding normal ovarian tissues. (B) Summarization of the PCR results. The 100% stacked column charts were used to compare the case numbers of differentially-expressed mRNAs in the ovarian cancer tissues. The total case numbers of differentially-expressed mRNAs (increased, decreased and no change) in the ovarian cancer tissues is equal to 100%. (C) Statistical analysis of quantified mRNA levels between the ovarian cancer and normal tissues. The mRNA expression signals shown in (A) were quantified by densitometry using Quantity One Basic Software. The significant difference is expressed as * P
    Figure Legend Snippet: A reduction in hMOF mRNA levels is observed in human ovarian cancer. (A) PCR analysis of 47 clinical ovarian cancer tissues. Total RNA was isolated from the tissues using TRIzol. The PCR assay was performed to detect the mRNA expression levels of hMOF, CA9, VEGF, HIF1α and hSTC1 in clinical ovarian cancer and normal ovarian tissues. The PCR products were then separated by electrophoresis on a 2% agarose gel. The DNA fragments were visualized and photographed under ultraviolet light with ethidium bromide. The mRNA levels from 37 ovarian cancer tissues were compared with corresponding contralateral ovarian normal tissues. However, 10 clinical ovarian cancer tissues were missing contralateral ovarian normal tissues and were compared with non-corresponding normal ovarian tissues. (B) Summarization of the PCR results. The 100% stacked column charts were used to compare the case numbers of differentially-expressed mRNAs in the ovarian cancer tissues. The total case numbers of differentially-expressed mRNAs (increased, decreased and no change) in the ovarian cancer tissues is equal to 100%. (C) Statistical analysis of quantified mRNA levels between the ovarian cancer and normal tissues. The mRNA expression signals shown in (A) were quantified by densitometry using Quantity One Basic Software. The significant difference is expressed as * P

    Techniques Used: Polymerase Chain Reaction, Isolation, Expressing, Electrophoresis, Agarose Gel Electrophoresis, Software

    7) Product Images from "RIG-I-like receptor activation drives type I IFN and antiviral signaling to limit Hantaan orthohantavirus replication"

    Article Title: RIG-I-like receptor activation drives type I IFN and antiviral signaling to limit Hantaan orthohantavirus replication

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008483

    Type I IFN required for early control of HTNV replication and dissemination in vivo . C57BL/6J (WT), Mavs -/- and Ifnar1 -/- mice were infected via intraperitoneal injection with 1x10 6 FFU HTNV 76–118 or mock infected with PBS. Mice (n = 3/genotype) were euthanized on days 3, 5, 7, and 14 post-infection. Whole lung, spleen, and kidney tissues were collected in 0.5% BSA in PBS and homogenized. 25% of each homogenized tissue was subjected to TRIzol RNA extraction. (A) Animal weight and clinical scores for all WT (mock in black, virus infected in blue), Ifnar1 -/- (mock in black, virus infected in red), and Mavs -/- (mock in black, virus infected in green). (B) Viral RNA was quantified by qRT-PCR and back-calculated as virus RNA copies/tissue (±SD). Each in vivo experiment was performed twice yielding n = 6 HTNV infected mice per genotype, per time point. Statistical significance determined by multiple T-tests using Holm-Sidak method, with alpha = 0.05, using Prism 8 software (* denotes p adjusted
    Figure Legend Snippet: Type I IFN required for early control of HTNV replication and dissemination in vivo . C57BL/6J (WT), Mavs -/- and Ifnar1 -/- mice were infected via intraperitoneal injection with 1x10 6 FFU HTNV 76–118 or mock infected with PBS. Mice (n = 3/genotype) were euthanized on days 3, 5, 7, and 14 post-infection. Whole lung, spleen, and kidney tissues were collected in 0.5% BSA in PBS and homogenized. 25% of each homogenized tissue was subjected to TRIzol RNA extraction. (A) Animal weight and clinical scores for all WT (mock in black, virus infected in blue), Ifnar1 -/- (mock in black, virus infected in red), and Mavs -/- (mock in black, virus infected in green). (B) Viral RNA was quantified by qRT-PCR and back-calculated as virus RNA copies/tissue (±SD). Each in vivo experiment was performed twice yielding n = 6 HTNV infected mice per genotype, per time point. Statistical significance determined by multiple T-tests using Holm-Sidak method, with alpha = 0.05, using Prism 8 software (* denotes p adjusted

    Techniques Used: In Vivo, Mouse Assay, Infection, Injection, RNA Extraction, Quantitative RT-PCR, Software

    8) Product Images from "The C-Terminal ?-Helix Domain of Apolipoprotein E Is Required for Interaction with Nonstructural Protein 5A and Assembly of Hepatitis C Virus ▿"

    Article Title: The C-Terminal ?-Helix Domain of Apolipoprotein E Is Required for Interaction with Nonstructural Protein 5A and Assembly of Hepatitis C Virus ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01021-10

    Determination of the effects of apoE deletion mutations on HCV production. (A) Quantification of HCV vRNA in the media by a real-time RT-PCR method. HCV vRNA in the media was extracted with Trizol LS reagent (Invitrogen). The levels of HCV vRNA were determined
    Figure Legend Snippet: Determination of the effects of apoE deletion mutations on HCV production. (A) Quantification of HCV vRNA in the media by a real-time RT-PCR method. HCV vRNA in the media was extracted with Trizol LS reagent (Invitrogen). The levels of HCV vRNA were determined

    Techniques Used: Quantitative RT-PCR

    9) Product Images from "Alterations in the host transcriptome in vitro following Rift Valley fever virus infection"

    Article Title: Alterations in the host transcriptome in vitro following Rift Valley fever virus infection

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14800-3

    Analysis of differentially expressed genes in HSAECs following RVFV infection. ( a ) Venn diagrams depict the upregulated (top panel) and downregulated (bottom panel) genes changed in MP12 only (left), ZH548 only (right), or both (center, gray) at 3, 9, and 18 hours post infection. These genes were changed by 1.5-fold or more and had a p-value ≤ 0.05. ( b ) HSAECs were mock-infected or infected with MP12 or ZH548 at MOI 5 for one hour. Lysates were collected in Trizol LS, RNA was extracted and prepared for RNA sequencing. RNA-sequencing reads were normalized to the total reads, then fold changes were calculated against the uninfected, mock samples at the specified time point. ( c ) RT-qPCR confirmation of some of the top changed transcripts during all time points post infection. HSAECs were mock-infected or infected with MP12 or ZH548 at MOI 5 for one hour. Lysates were collected in Trizol LS, RNA was extracted using the Direct-zol™ RNA MiniPrep, and analyzed for RT-qPCR with TaqMan Gene Expression Assays against IFIT1, IFIT2, IFIT3, and RSAD2. Fold changes were calculated relative to 18 S ribosomal RNA and normalized to mock samples using the ΔΔCt method. Data are expressed as the Mean ± SD (n = 3).
    Figure Legend Snippet: Analysis of differentially expressed genes in HSAECs following RVFV infection. ( a ) Venn diagrams depict the upregulated (top panel) and downregulated (bottom panel) genes changed in MP12 only (left), ZH548 only (right), or both (center, gray) at 3, 9, and 18 hours post infection. These genes were changed by 1.5-fold or more and had a p-value ≤ 0.05. ( b ) HSAECs were mock-infected or infected with MP12 or ZH548 at MOI 5 for one hour. Lysates were collected in Trizol LS, RNA was extracted and prepared for RNA sequencing. RNA-sequencing reads were normalized to the total reads, then fold changes were calculated against the uninfected, mock samples at the specified time point. ( c ) RT-qPCR confirmation of some of the top changed transcripts during all time points post infection. HSAECs were mock-infected or infected with MP12 or ZH548 at MOI 5 for one hour. Lysates were collected in Trizol LS, RNA was extracted using the Direct-zol™ RNA MiniPrep, and analyzed for RT-qPCR with TaqMan Gene Expression Assays against IFIT1, IFIT2, IFIT3, and RSAD2. Fold changes were calculated relative to 18 S ribosomal RNA and normalized to mock samples using the ΔΔCt method. Data are expressed as the Mean ± SD (n = 3).

    Techniques Used: Infection, RNA Sequencing Assay, Quantitative RT-PCR, Expressing

    10) Product Images from "Epigenetic change in kidney tumor: downregulation of histone acetyltransferase MYST1 in human renal cell carcinoma"

    Article Title: Epigenetic change in kidney tumor: downregulation of histone acetyltransferase MYST1 in human renal cell carcinoma

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-32-8

    hMOF is downregulated in human ccRCC. A. Clinical informations of four newly diagnosed patients with ccRCC. B. hMOF mRNA levels are dropped down in 4 random cases of ccRCC tissues. Total RNA from tissue was isolated using trizol. mRNA levels of hMOF, CA9, VEGF and HIF1α in paired human clinical ccRCC and adjacent kidney tissue was analyzed by RT-PCR (upper panel). mRNA levels were quantified by densitometry using Quantity One Basic software (Bio- Rad) (lower panel). C. Total hMOF protein expression and the acetylation of histone H4K16 levels are decreased in selected ccRCC tumor tissue. Aliquots of whole cell extracts from four selected ccRCC tumor samples and its corresponding adjacent tissues were subjected to SDS-PAGE in 12% gels, and proteins were detected by western blotting with indicated antibodies (upper panel). Western blot images were quantified using Quantity One software (Bio-Rad) (lower panel). The significant difference is expressed as *p
    Figure Legend Snippet: hMOF is downregulated in human ccRCC. A. Clinical informations of four newly diagnosed patients with ccRCC. B. hMOF mRNA levels are dropped down in 4 random cases of ccRCC tissues. Total RNA from tissue was isolated using trizol. mRNA levels of hMOF, CA9, VEGF and HIF1α in paired human clinical ccRCC and adjacent kidney tissue was analyzed by RT-PCR (upper panel). mRNA levels were quantified by densitometry using Quantity One Basic software (Bio- Rad) (lower panel). C. Total hMOF protein expression and the acetylation of histone H4K16 levels are decreased in selected ccRCC tumor tissue. Aliquots of whole cell extracts from four selected ccRCC tumor samples and its corresponding adjacent tissues were subjected to SDS-PAGE in 12% gels, and proteins were detected by western blotting with indicated antibodies (upper panel). Western blot images were quantified using Quantity One software (Bio-Rad) (lower panel). The significant difference is expressed as *p

    Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Software, Expressing, SDS Page, Western Blot

    Non-correlation between hMOF and CA9 is found in renal cell carcinoma cells. A. hMOF protein expression was correlated with acetylation of H4K16 in RCC cell 786–0 and OSRC-2. 293T, 786–0 or OSRC-2 cells were cultured in 6-well tissue culture plates (~2x10 5 cells/well) in DMEM medium containing 10% fetal bovine serum. Whole cell extracts were subjected to immunoblotting using indicated antibodies (right panel). 293T, 786–0 or OSRC-2 cells from 1 well of a 6 well plate were lysed and total RNA was isolated using Trizol. hMOF and CA9 gene expressions were measured by RT-PCR (left panel) and qRT-PCR ( B ). C. Effect of hMOF on CA9 mRNA expression levels in RCC cells. RCC 786–0 cells were cultured in 6-well tissue culture plates (~2x10 5 cells/well) in DMEM medium containing 10% fetal bovine serum. The cells were transfected with 0.25, 0.5, 1 and 2 μg of hMOF cDNAs. 48 hours after transfection, cells were lysed and total RNA was isolated using Trizol. Indicated gene expressions were analyzed by qRT-PCR. D. Effect of hMOF on CA9 protein expression in RCC cells. RCC 786–0 cells were transfected with 0.25, 0.5, 1 and 2 μg of hMOF cDNAs. 48 hours after transfection, cells were harvested and lysed in RIPA buffer. Aliquots of whole cell extracts were subjected to 12% SDS-PAGE, and specific proteins were detected by indicated antibodies.
    Figure Legend Snippet: Non-correlation between hMOF and CA9 is found in renal cell carcinoma cells. A. hMOF protein expression was correlated with acetylation of H4K16 in RCC cell 786–0 and OSRC-2. 293T, 786–0 or OSRC-2 cells were cultured in 6-well tissue culture plates (~2x10 5 cells/well) in DMEM medium containing 10% fetal bovine serum. Whole cell extracts were subjected to immunoblotting using indicated antibodies (right panel). 293T, 786–0 or OSRC-2 cells from 1 well of a 6 well plate were lysed and total RNA was isolated using Trizol. hMOF and CA9 gene expressions were measured by RT-PCR (left panel) and qRT-PCR ( B ). C. Effect of hMOF on CA9 mRNA expression levels in RCC cells. RCC 786–0 cells were cultured in 6-well tissue culture plates (~2x10 5 cells/well) in DMEM medium containing 10% fetal bovine serum. The cells were transfected with 0.25, 0.5, 1 and 2 μg of hMOF cDNAs. 48 hours after transfection, cells were lysed and total RNA was isolated using Trizol. Indicated gene expressions were analyzed by qRT-PCR. D. Effect of hMOF on CA9 protein expression in RCC cells. RCC 786–0 cells were transfected with 0.25, 0.5, 1 and 2 μg of hMOF cDNAs. 48 hours after transfection, cells were harvested and lysed in RIPA buffer. Aliquots of whole cell extracts were subjected to 12% SDS-PAGE, and specific proteins were detected by indicated antibodies.

    Techniques Used: Expressing, Cell Culture, Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, SDS Page

    11) Product Images from "A potential diagnostic marker for ovarian cancer: Involvement of the histone acetyltransferase, human males absent on the first"

    Article Title: A potential diagnostic marker for ovarian cancer: Involvement of the histone acetyltransferase, human males absent on the first

    Journal: Oncology Letters

    doi: 10.3892/ol.2013.1380

    A reduction in hMOF mRNA levels is observed in human ovarian cancer. (A) PCR analysis of 47 clinical ovarian cancer tissues. Total RNA was isolated from the tissues using TRIzol. The PCR assay was performed to detect the mRNA expression levels of hMOF, CA9, VEGF, HIF1α and hSTC1 in clinical ovarian cancer and normal ovarian tissues. The PCR products were then separated by electrophoresis on a 2% agarose gel. The DNA fragments were visualized and photographed under ultraviolet light with ethidium bromide. The mRNA levels from 37 ovarian cancer tissues were compared with corresponding contralateral ovarian normal tissues. However, 10 clinical ovarian cancer tissues were missing contralateral ovarian normal tissues and were compared with non-corresponding normal ovarian tissues. (B) Summarization of the PCR results. The 100% stacked column charts were used to compare the case numbers of differentially-expressed mRNAs in the ovarian cancer tissues. The total case numbers of differentially-expressed mRNAs (increased, decreased and no change) in the ovarian cancer tissues is equal to 100%. (C) Statistical analysis of quantified mRNA levels between the ovarian cancer and normal tissues. The mRNA expression signals shown in (A) were quantified by densitometry using Quantity One Basic Software. The significant difference is expressed as * P
    Figure Legend Snippet: A reduction in hMOF mRNA levels is observed in human ovarian cancer. (A) PCR analysis of 47 clinical ovarian cancer tissues. Total RNA was isolated from the tissues using TRIzol. The PCR assay was performed to detect the mRNA expression levels of hMOF, CA9, VEGF, HIF1α and hSTC1 in clinical ovarian cancer and normal ovarian tissues. The PCR products were then separated by electrophoresis on a 2% agarose gel. The DNA fragments were visualized and photographed under ultraviolet light with ethidium bromide. The mRNA levels from 37 ovarian cancer tissues were compared with corresponding contralateral ovarian normal tissues. However, 10 clinical ovarian cancer tissues were missing contralateral ovarian normal tissues and were compared with non-corresponding normal ovarian tissues. (B) Summarization of the PCR results. The 100% stacked column charts were used to compare the case numbers of differentially-expressed mRNAs in the ovarian cancer tissues. The total case numbers of differentially-expressed mRNAs (increased, decreased and no change) in the ovarian cancer tissues is equal to 100%. (C) Statistical analysis of quantified mRNA levels between the ovarian cancer and normal tissues. The mRNA expression signals shown in (A) were quantified by densitometry using Quantity One Basic Software. The significant difference is expressed as * P

    Techniques Used: Polymerase Chain Reaction, Isolation, Expressing, Electrophoresis, Agarose Gel Electrophoresis, Software

    12) Product Images from "Cells release subpopulations of exosomes with distinct molecular and biological properties"

    Article Title: Cells release subpopulations of exosomes with distinct molecular and biological properties

    Journal: Scientific Reports

    doi: 10.1038/srep22519

    EV subpopulations have different RNA profiles. RNA from MV, LD-Exo and HD-Exo was extracted using Trizol and analyzed using capillary electrophoresis with the Agilent RNA 6000 Pico chip (left panel) and Agilent small RNA chip (right panel) on an Agilent 2100 Bioanalyzer®. The y-axis of the electropherograms represents fluorescence units (FU) and the x-axis represents the nucleotide length of the RNA (nt). Peaks at 25 nt (left panels) or at 4 nt (right panels) represent internal standards. Data shown are representative of two independent experiments.
    Figure Legend Snippet: EV subpopulations have different RNA profiles. RNA from MV, LD-Exo and HD-Exo was extracted using Trizol and analyzed using capillary electrophoresis with the Agilent RNA 6000 Pico chip (left panel) and Agilent small RNA chip (right panel) on an Agilent 2100 Bioanalyzer®. The y-axis of the electropherograms represents fluorescence units (FU) and the x-axis represents the nucleotide length of the RNA (nt). Peaks at 25 nt (left panels) or at 4 nt (right panels) represent internal standards. Data shown are representative of two independent experiments.

    Techniques Used: Electrophoresis, Chromatin Immunoprecipitation, Fluorescence

    13) Product Images from "Rapid and scalable profiling of nascent RNA with fastGRO"

    Article Title: Rapid and scalable profiling of nascent RNA with fastGRO

    Journal: bioRxiv

    doi: 10.1101/2020.01.24.916015

    Overview of fastGRO. In day 1, nuclei are isolated and in vitro run-on is performed in a solution containing 4-thio-UTP that is incorporated in nascent RNA. After isolation using Trizol and ethanol precipitation, RNA is fragmented and snap-frozen. In day 2, 4-thio-UTP containing RNA is biotinylated using either HPDP- (standard protocol) or MTS-biotin (fastGRO-LI protocol for low input sample) and recovered by immunoprecipitation using streptavidin-conjugated beads. Labeled RNA is recovered by elution in DTT solution, purified and used for NGS libraries preparation with commercially available kits.
    Figure Legend Snippet: Overview of fastGRO. In day 1, nuclei are isolated and in vitro run-on is performed in a solution containing 4-thio-UTP that is incorporated in nascent RNA. After isolation using Trizol and ethanol precipitation, RNA is fragmented and snap-frozen. In day 2, 4-thio-UTP containing RNA is biotinylated using either HPDP- (standard protocol) or MTS-biotin (fastGRO-LI protocol for low input sample) and recovered by immunoprecipitation using streptavidin-conjugated beads. Labeled RNA is recovered by elution in DTT solution, purified and used for NGS libraries preparation with commercially available kits.

    Techniques Used: Isolation, In Vitro, Ethanol Precipitation, Immunoprecipitation, Labeling, Purification, Next-Generation Sequencing

    14) 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

    15) Product Images from "Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods"

    Article Title: Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-018-1651-z

    RNA quantification. a Bar graph of total RNA quantified from FHHNC uEVs by Bioanalyzer—Picochip using the five different extraction methods. MirCURY kit followed by TRIzol LS were the most efficient methods. b A representative electropherogram shows that uEVs contain small RNA, including microRNAs (10–40 nt). As expected, small amounts of rRNA were detected. c miRNA profiling by RT-qPCR of RNA extracted from FHHNC uEVs. miRNA expression pattern is consistent independently of the RNA extraction method
    Figure Legend Snippet: RNA quantification. a Bar graph of total RNA quantified from FHHNC uEVs by Bioanalyzer—Picochip using the five different extraction methods. MirCURY kit followed by TRIzol LS were the most efficient methods. b A representative electropherogram shows that uEVs contain small RNA, including microRNAs (10–40 nt). As expected, small amounts of rRNA were detected. c miRNA profiling by RT-qPCR of RNA extracted from FHHNC uEVs. miRNA expression pattern is consistent independently of the RNA extraction method

    Techniques Used: Quantitative RT-PCR, Expressing, RNA Extraction

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

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

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2017.1401897

    The bulk of milk microRNA pellets at low ultracentrifugation speeds and microRNAs distribution do not correspond to EV-associated proteins profiles. (a) Commercial milk preparation (100 mL) was subjected to successive differential ultracentrifugation steps at 12,000 g , 35,000 g , 70,000 g and 100,000 g for 1 h each at 4°C, and each pellet was kept and suspended in 1 mL PBS containing EDTA, as detailed in the Material and methods section. (b) Each sample (250 µL) was mixed with 750 µL of TRIzol-LS reagent for total RNA isolation and subsequent RT-qPCR detection of microRNAs bta-miR-223, bta-miR-125b, bta-miR-148a, bta-miR-29b, bta-miR-151-3p and bta-miR-2478. The experiment was performed three times with three different milk samples, and each quantification level in each pellet was reported on the sum of all the pellet (total) and expressed as a percentage of the total (mean ± SD; n = 3 or 6). The statistical significance of the differences observed was assessed by an RM one-way ANOVA with Geisser–Greenhouse correction coupled with a post hoc comparison of the means with Tukey’s correction with p
    Figure Legend Snippet: The bulk of milk microRNA pellets at low ultracentrifugation speeds and microRNAs distribution do not correspond to EV-associated proteins profiles. (a) Commercial milk preparation (100 mL) was subjected to successive differential ultracentrifugation steps at 12,000 g , 35,000 g , 70,000 g and 100,000 g for 1 h each at 4°C, and each pellet was kept and suspended in 1 mL PBS containing EDTA, as detailed in the Material and methods section. (b) Each sample (250 µL) was mixed with 750 µL of TRIzol-LS reagent for total RNA isolation and subsequent RT-qPCR detection of microRNAs bta-miR-223, bta-miR-125b, bta-miR-148a, bta-miR-29b, bta-miR-151-3p and bta-miR-2478. The experiment was performed three times with three different milk samples, and each quantification level in each pellet was reported on the sum of all the pellet (total) and expressed as a percentage of the total (mean ± SD; n = 3 or 6). The statistical significance of the differences observed was assessed by an RM one-way ANOVA with Geisser–Greenhouse correction coupled with a post hoc comparison of the means with Tukey’s correction with p

    Techniques Used: Isolation, Quantitative RT-PCR

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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

    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

    (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

    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