trizol ls reagent  (Thermo Fisher)


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    TRIzol Reagent
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    Thermo Fisher trizol ls reagent
    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 <t>Trizol</t> ® LS, <t>miRNeasy</t> ® , 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

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    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    6) 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

    7) 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

    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:

    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 "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

    10) 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

    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

    Related Articles

    Amplification:

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    Article Snippet: Total RNAs in leucocytes and plasma were extracted with TRIzol and TRIzol LS (Invitogen; Thermo Fisher Scientific, Inc.), respectively by following the manufacturer's protocol. .. Total RNAs in leucocytes and plasma were extracted with TRIzol and TRIzol LS (Invitogen; Thermo Fisher Scientific, Inc.), respectively by following the manufacturer's protocol.

    Article Title: Silencing long non-coding RNA Kcnq1ot1 alleviates pyroptosis and fibrosis in diabetic cardiomyopathy
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    Article Snippet: Paragraph title: Single-genome amplification. ... Total RNA was isolated from blood and semen plasma and infected cells using TRIzol-LS and TRIzol, respectively, according to the manufacturer's instructions (Invitrogen).

    Synthesized:

    Article Title: miR-375 Modulates Radiosensitivity of HR-HPV-Positive Cervical Cancer Cells by Targeting UBE3A through the p53 Pathway
    Article Snippet: HEK 293T, HPV-16-positive SiHa and HPV-18-positive HeLa cervical cancer cell lines were obtained from the ATCC and were cultured with Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories), 2 mM/L-glutamine, 100U/mL penicillin, and 100 mg/mL streptomycin and were incubated in humidified air with 5% CO2 at 37°C. .. Total RNAs from tissue and cells were extracted using TRIzol reagent (Invitrogen) and from blood were extracted using TRIzol LS reagent (Invitrogen), respectively. cDNAs were synthesized using the PrimeScript RT reagent Kit (TaKaRa). miR-375 expression was quantified by using TaqMan MicroRNA assays (Life Technologies). .. The 2−ΔΔ Ct method was used to calculate relative miR-375 expression, with RNU6B as a control gene.

    Telomerase Activity Assay:

    Article Title: Circulating and tissue biomarkers in early-stage non-small cell lung cancer
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    Real-time Polymerase Chain Reaction:

    Article Title: Stearoly-CoA desaturase 1 differentiates early and advanced dengue virus infections and determines virus particle infectivity
    Article Snippet: RNA was extracted from cells using Trizol (ThermoFisher) and from virus in supernatant using Trizol LS (ThermoFisher). .. A one-step qRT-PCR kit with SYBR green from Agilent was used.

    Article Title: Clinical Characterization of Host Response to Simian Hemorrhagic Fever Virus Infection in Permissive and Refractory Hosts: A Model for Determining Mechanisms of VHF Pathogenesis
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    Article Title: Circulating and tissue biomarkers in early-stage non-small cell lung cancer
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    Article Title: Skin adipocyte stem cell self-renewal is regulated by a Pdgfa/Akt signaling axis
    Article Snippet: Paragraph title: RNA extraction and Real-Time PCR ... APs were either sorted directly into Trizol LS (Invitrogen 10296-028) or lysed in 24 well plates by adding Trizol (Invitrogen 15596-026).

    Incubation:

    Article Title: LncRNA KCNQ1OT1 regulates proliferation and cisplatin resistance in tongue cancer via miR-211-5p mediated Ezrin/Fak/Src signaling
    Article Snippet: Cells (1 × 107 ) were collected, resuspended in 1 ml of ice-cold cell fractionation buffer and incubated for 15 min on ice. .. Then, the cells were centrifuged for 15 min at 500 g ; the supernatant and nuclear pellet were reserved for RNA extraction using TRIzol LS and TRIzol reagent (Invitrogen, USA) using a previously established protocol .

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    Cell Culture:

    Article Title: Characterization of Haartman Institute snake virus-1 (HISV-1) and HISV-like viruses—The representatives of genus Hartmanivirus, family Arenaviridae
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    Expressing:

    Article Title: CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo
    Article Snippet: Supernatant and cell associated RNA was extracted using TRIzol LS and TRizol reagents, respectively according to the manufacturer’s protocols (Invitrogen) and converted to cDNA using qScript cDNA Supermix or qScript Flex cDNA Kit according to manufacturer’s instructions (Quanta Biosciences). .. RNA from viral supernatants was quantified by real time PCR using TaqMan Fast Mastermix (Applied Biosystems) on an Applied Biosystems 7300 thermocycler using primers and probes as previously described [ ] and used in SGA PCR described below.

    Article Title: miR-375 Modulates Radiosensitivity of HR-HPV-Positive Cervical Cancer Cells by Targeting UBE3A through the p53 Pathway
    Article Snippet: HEK 293T, HPV-16-positive SiHa and HPV-18-positive HeLa cervical cancer cell lines were obtained from the ATCC and were cultured with Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories), 2 mM/L-glutamine, 100U/mL penicillin, and 100 mg/mL streptomycin and were incubated in humidified air with 5% CO2 at 37°C. .. Total RNAs from tissue and cells were extracted using TRIzol reagent (Invitrogen) and from blood were extracted using TRIzol LS reagent (Invitrogen), respectively. cDNAs were synthesized using the PrimeScript RT reagent Kit (TaKaRa). miR-375 expression was quantified by using TaqMan MicroRNA assays (Life Technologies). .. The 2−ΔΔ Ct method was used to calculate relative miR-375 expression, with RNU6B as a control gene.

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    Cell Fractionation:

    Article Title: LncRNA KCNQ1OT1 regulates proliferation and cisplatin resistance in tongue cancer via miR-211-5p mediated Ezrin/Fak/Src signaling
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    Modification:

    Article Title: Alternative splicing events expand molecular diversity of camel CSN1S2 increasing its ability to generate potentially bioactive peptides
    Article Snippet: The most representative eight milk samples (C . bactrianus , n = 3, C . dromedarius , n = 3, and hybrids, n = 2) were analyzed by LC-MS/MS (LTQ-Orbitrap Discovery, Thermo Fisher Scientific) after a tryptic digestion of bands, excised from each track, between 20 and 30 kDa of SDS-PAGE. .. Total RNA was extracted from MFG using TRIzol® and TRIzol® LS solutions (Invitrogen, Life Technologies), respectively, according to the original manufacturer’s protocol modified as described by Brenaut et al . .. First-strand cDNA was synthesized from 5 to 10 ng of total RNA primed with oligo(dT)20 and random primers (3:1, vol/vol) using Superscript III reverse transcriptase (Invitrogen Life Technologies Inc., Carlsbad, CA) as described previously .

    Sequencing:

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    Biomarker Assay:

    Article Title: Circulating and tissue biomarkers in early-stage non-small cell lung cancer
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    Infection:

    Article Title: Seminal Simian Immunodeficiency Virus in Chronically Infected Cynomolgus Macaques Is Dominated by Virus Originating from Multiple Genital Organs
    Article Snippet: Given that both cell-associated and cell-free viruses are present in semen, and that it is currently not clear which of these initiates a new infection, we chose to amplify both cell-associated and free virus in semen. .. Total RNA was isolated from blood and semen plasma and infected cells using TRIzol-LS and TRIzol, respectively, according to the manufacturer's instructions (Invitrogen). .. Genital tract tissue samples were homogenized with a TissuLyser II system (Qiagen), and total RNA was extracted using TRIzol (Invitrogen) following the manufacturer's suggested protocol.

    Polymerase Chain Reaction:

    Article Title: Genetic Variants in Human Leukocyte Antigen-DP Influence Both Hepatitis C Virus Persistence and Hepatitis C Virus F Protein Generation in the Chinese Han Population
    Article Snippet: Anti-HCV antibodies were tested by the third generation HCV enzyme immunoassays (KHB, Shanghai, China). .. HCV RNA was extracted from serum using Trizol reagent (Trizol LS Reagent, Life Technologies, Rockville, MD, USA) and detected by polymerase chain reaction (Realchip Biotechnology Co., Ltd., Ningbo, China) according to the manufacturer’s instructions. .. The reaction products were detected in a 2% agarose gel (Biowest Agarose, Hong Kong, China) stained with Gel stain (Beijing Transgen Biotech Co., Ltd., Beijing, China).

    Article Title: miR-375 Modulates Radiosensitivity of HR-HPV-Positive Cervical Cancer Cells by Targeting UBE3A through the p53 Pathway
    Article Snippet: Total RNAs from tissue and cells were extracted using TRIzol reagent (Invitrogen) and from blood were extracted using TRIzol LS reagent (Invitrogen), respectively. cDNAs were synthesized using the PrimeScript RT reagent Kit (TaKaRa). miR-375 expression was quantified by using TaqMan MicroRNA assays (Life Technologies). .. The 2−ΔΔ Ct method was used to calculate relative miR-375 expression, with RNU6B as a control gene.

    Article Title: Circulating and tissue biomarkers in early-stage non-small cell lung cancer
    Article Snippet: Briefly, total RNA purification, including miRNAs, was based on lysis with guanidinium thiocyanate-phenol-chloroform extraction (TriZol-LS, Applied Biosystem) and Spin Column-based total RNA purification (MiRneasy Mini Kit, Qiagen). .. Briefly, total RNA purification, including miRNAs, was based on lysis with guanidinium thiocyanate-phenol-chloroform extraction (TriZol-LS, Applied Biosystem) and Spin Column-based total RNA purification (MiRneasy Mini Kit, Qiagen).

    Recombinant:

    Article Title: Seminal Simian Immunodeficiency Virus in Chronically Infected Cynomolgus Macaques Is Dominated by Virus Originating from Multiple Genital Organs
    Article Snippet: Total RNA was isolated from blood and semen plasma and infected cells using TRIzol-LS and TRIzol, respectively, according to the manufacturer's instructions (Invitrogen). .. Total RNA was isolated from blood and semen plasma and infected cells using TRIzol-LS and TRIzol, respectively, according to the manufacturer's instructions (Invitrogen).

    Enzyme Immunoassay:

    Article Title: Genetic Variants in Human Leukocyte Antigen-DP Influence Both Hepatitis C Virus Persistence and Hepatitis C Virus F Protein Generation in the Chinese Han Population
    Article Snippet: Anti-HCV antibodies were tested by the third generation HCV enzyme immunoassays (KHB, Shanghai, China). .. HCV RNA was extracted from serum using Trizol reagent (Trizol LS Reagent, Life Technologies, Rockville, MD, USA) and detected by polymerase chain reaction (Realchip Biotechnology Co., Ltd., Ningbo, China) according to the manufacturer’s instructions.

    Isolation:

    Article Title: A Novel Actin mRNA Splice Variant Regulates ACTG1 Expression
    Article Snippet: Paragraph title: Nuclear and cytoplasmic RNA isolation ... The cytosol-containing supernatant was examined under the microscope to assure the absence of nuclei in the cytosolic fraction prior to the addition of TRIzol LS to the supernatant and TRIzol to the pellet (Invitrogen, Carlsbad, CA).

    Article Title: Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix
    Article Snippet: The use of different RNA extraction kits – may affect the miRNA recovery from serum dry-spotted on pre-treated FTA Elute cards. .. To address this, we compared the performance of QIAzol lysis reagent with Trizol-LS reagent, Trizol reagent and column-based RNA isolation kit (Ambion). .. Our results showed that the average percentage miRNA recovery of all 10 miRNAs across these methods was similar (Supplementary Fig. ).

    Article Title: Characterization of Haartman Institute snake virus-1 (HISV-1) and HISV-like viruses—The representatives of genus Hartmanivirus, family Arenaviridae
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    Article Title: CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo
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    Article Title: A revised airway epithelial hierarchy includes CFTR-expressing ionocytes
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    Article Title: Silencing long non-coding RNA Kcnq1ot1 alleviates pyroptosis and fibrosis in diabetic cardiomyopathy
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    Article Title: Seminal Simian Immunodeficiency Virus in Chronically Infected Cynomolgus Macaques Is Dominated by Virus Originating from Multiple Genital Organs
    Article Snippet: Given that both cell-associated and cell-free viruses are present in semen, and that it is currently not clear which of these initiates a new infection, we chose to amplify both cell-associated and free virus in semen. .. Total RNA was isolated from blood and semen plasma and infected cells using TRIzol-LS and TRIzol, respectively, according to the manufacturer's instructions (Invitrogen). .. Genital tract tissue samples were homogenized with a TissuLyser II system (Qiagen), and total RNA was extracted using TRIzol (Invitrogen) following the manufacturer's suggested protocol.

    Article Title: The altered expression of inflammation-related microRNAs with microRNA-155 expression correlates with Th17 differentiation in patients with acute coronary syndrome
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    Microscopy:

    Article Title: A Novel Actin mRNA Splice Variant Regulates ACTG1 Expression
    Article Snippet: Samples were centrifuged at 1,000×g for 5 minutes to pellet nuclei. .. The cytosol-containing supernatant was examined under the microscope to assure the absence of nuclei in the cytosolic fraction prior to the addition of TRIzol LS to the supernatant and TRIzol to the pellet (Invitrogen, Carlsbad, CA). .. RNA purification of both the cytosolic and nuclear fractions was done according to manufacturer's instructions.

    Purification:

    Article Title: Genetic Variants in Human Leukocyte Antigen-DP Influence Both Hepatitis C Virus Persistence and Hepatitis C Virus F Protein Generation in the Chinese Han Population
    Article Snippet: HCV RNA was extracted from serum using Trizol reagent (Trizol LS Reagent, Life Technologies, Rockville, MD, USA) and detected by polymerase chain reaction (Realchip Biotechnology Co., Ltd., Ningbo, China) according to the manufacturer’s instructions. .. HCV RNA was extracted from serum using Trizol reagent (Trizol LS Reagent, Life Technologies, Rockville, MD, USA) and detected by polymerase chain reaction (Realchip Biotechnology Co., Ltd., Ningbo, China) according to the manufacturer’s instructions.

    Article Title: Circulating and tissue biomarkers in early-stage non-small cell lung cancer
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    Indirect ELISA:

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    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Characterization of Haartman Institute snake virus-1 (HISV-1) and HISV-like viruses—The representatives of genus Hartmanivirus, family Arenaviridae
    Article Snippet: Trizol and Trizol LS isolation reagent (Life Technologies) in combination with QiaGEN RNeasy Mini Kit (Qiagen) was used for RNA isolation as described [ ]. .. No carrier RNA was used during RNA isolation for samples analyzed by NGS.

    Article Title: Clinical Characterization of Host Response to Simian Hemorrhagic Fever Virus Infection in Permissive and Refractory Hosts: A Model for Determining Mechanisms of VHF Pathogenesis
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    Quantitative RT-PCR:

    Article Title: Stearoly-CoA desaturase 1 differentiates early and advanced dengue virus infections and determines virus particle infectivity
    Article Snippet: Paragraph title: RNA extraction and qRT-PCR ... RNA was extracted from cells using Trizol (ThermoFisher) and from virus in supernatant using Trizol LS (ThermoFisher).

    Article Title: miR-375 Modulates Radiosensitivity of HR-HPV-Positive Cervical Cancer Cells by Targeting UBE3A through the p53 Pathway
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    Article Title: A revised airway epithelial hierarchy includes CFTR-expressing ionocytes
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    Article Title: Clinical Characterization of Host Response to Simian Hemorrhagic Fever Virus Infection in Permissive and Refractory Hosts: A Model for Determining Mechanisms of VHF Pathogenesis
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    Article Title: Silencing long non-coding RNA Kcnq1ot1 alleviates pyroptosis and fibrosis in diabetic cardiomyopathy
    Article Snippet: Paragraph title: Total RNA isolation and quantitative real-time RT-PCR (qRT-PCR) ... Total RNA in the human serums was extracted by Trizol LS (Invitrogen, USA), and the total RNA of cardiac fibroblasts and cardiac tissues was extracted by Trizol (Invitrogen, USA).

    Article Title: Circulating and tissue biomarkers in early-stage non-small cell lung cancer
    Article Snippet: Briefly, total RNA purification, including miRNAs, was based on lysis with guanidinium thiocyanate-phenol-chloroform extraction (TriZol-LS, Applied Biosystem) and Spin Column-based total RNA purification (MiRneasy Mini Kit, Qiagen). .. MiRNA qRT-PCR was carried out on a ViiA™ 7 instruments (ThermoFisher) using the manufacturer’s recommended cycling conditions.

    FACS:

    Article Title: A revised airway epithelial hierarchy includes CFTR-expressing ionocytes
    Article Snippet: Libraries were sequenced on an Illumina Nextseq 500. .. FACS isolated cells were sorted into 150 µl TRIzol LS (ThermoFisher Scientific), while ALI culture membranes were submerged in 300 µl of standard TRIzol solution (ThermoFisher Scientific). .. A standard chloroform extraction was performed followed by an RNeasy column-based RNA purification (Qiagen) according to manufacturer’s instructions.

    SYBR Green Assay:

    Article Title: miR-375 Modulates Radiosensitivity of HR-HPV-Positive Cervical Cancer Cells by Targeting UBE3A through the p53 Pathway
    Article Snippet: Total RNAs from tissue and cells were extracted using TRIzol reagent (Invitrogen) and from blood were extracted using TRIzol LS reagent (Invitrogen), respectively. cDNAs were synthesized using the PrimeScript RT reagent Kit (TaKaRa). miR-375 expression was quantified by using TaqMan MicroRNA assays (Life Technologies). .. The 2−ΔΔ Ct method was used to calculate relative miR-375 expression, with RNU6B as a control gene.

    Article Title: Skin adipocyte stem cell self-renewal is regulated by a Pdgfa/Akt signaling axis
    Article Snippet: APs were either sorted directly into Trizol LS (Invitrogen 10296-028) or lysed in 24 well plates by adding Trizol (Invitrogen 15596-026). .. RNA extraction and purification was done using the RNeasy mini kit (QIAGEN 74104) following manufacturer instructions. cDNA was generated using equal amounts of total RNA with the Superscript III First-Strand Synthesis System (Invitrogen 18080051) using Oligo dT per the manufacturer’s instructions.

    RNA Extraction:

    Article Title: Stearoly-CoA desaturase 1 differentiates early and advanced dengue virus infections and determines virus particle infectivity
    Article Snippet: Paragraph title: RNA extraction and qRT-PCR ... RNA was extracted from cells using Trizol (ThermoFisher) and from virus in supernatant using Trizol LS (ThermoFisher).

    Article Title: LncRNA KCNQ1OT1 regulates proliferation and cisplatin resistance in tongue cancer via miR-211-5p mediated Ezrin/Fak/Src signaling
    Article Snippet: Cells (1 × 107 ) were collected, resuspended in 1 ml of ice-cold cell fractionation buffer and incubated for 15 min on ice. .. Then, the cells were centrifuged for 15 min at 500 g ; the supernatant and nuclear pellet were reserved for RNA extraction using TRIzol LS and TRIzol reagent (Invitrogen, USA) using a previously established protocol . .. Total RNA was isolated from cells or tissues using TRIzol reagent (Invitrogen, USA) and then was converted to cDNA using an M-MLV Reverse Transcriptase Kit (Invitrogen, USA).

    Article Title: Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix
    Article Snippet: The use of different RNA extraction kits – may affect the miRNA recovery from serum dry-spotted on pre-treated FTA Elute cards. .. To address this, we compared the performance of QIAzol lysis reagent with Trizol-LS reagent, Trizol reagent and column-based RNA isolation kit (Ambion).

    Article Title: Screening of circular RNAs and validation of circANKRD36 associated with inflammation in patients with type 2 diabetes mellitus
    Article Snippet: Paragraph title: RNA extraction and RT ... Total RNAs in leucocytes and plasma were extracted with TRIzol and TRIzol LS (Invitogen; Thermo Fisher Scientific, Inc.), respectively by following the manufacturer's protocol.

    Article Title: Alternative splicing events expand molecular diversity of camel CSN1S2 increasing its ability to generate potentially bioactive peptides
    Article Snippet: Paragraph title: RNA Extraction from Milk Fat Globules ... Total RNA was extracted from MFG using TRIzol® and TRIzol® LS solutions (Invitrogen, Life Technologies), respectively, according to the original manufacturer’s protocol modified as described by Brenaut et al .

    Article Title: Skin adipocyte stem cell self-renewal is regulated by a Pdgfa/Akt signaling axis
    Article Snippet: Paragraph title: RNA extraction and Real-Time PCR ... APs were either sorted directly into Trizol LS (Invitrogen 10296-028) or lysed in 24 well plates by adding Trizol (Invitrogen 15596-026).

    Next-Generation Sequencing:

    Article Title: Characterization of Haartman Institute snake virus-1 (HISV-1) and HISV-like viruses—The representatives of genus Hartmanivirus, family Arenaviridae
    Article Snippet: Trizol and Trizol LS isolation reagent (Life Technologies) in combination with QiaGEN RNeasy Mini Kit (Qiagen) was used for RNA isolation as described [ ]. .. RNA isolation from cell culture supernatants was done with either the QIAamp Viral RNA Mini Kit (Qiagen) or the GeneJET RNA Purification Kit (Thermo Fisher Scientific) following the manufacturer’s instructions.

    Produced:

    Article Title: Seminal Simian Immunodeficiency Virus in Chronically Infected Cynomolgus Macaques Is Dominated by Virus Originating from Multiple Genital Organs
    Article Snippet: In order to maximize the chances of detecting viral sequences in cells and organs matching those of free virus in semen, we chose to focus on the viral strains being currently produced and therefore used viral RNA in tissues and cells for sequencing rather than viral DNA, which can represent archived/defective viruses. .. Total RNA was isolated from blood and semen plasma and infected cells using TRIzol-LS and TRIzol, respectively, according to the manufacturer's instructions (Invitrogen).

    Concentration Assay:

    Article Title: Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix
    Article Snippet: We next determined the effect of varying trehalose concentration for FTA Elute cards pre-treatment. .. To address this, we compared the performance of QIAzol lysis reagent with Trizol-LS reagent, Trizol reagent and column-based RNA isolation kit (Ambion).

    Article Title: The altered expression of inflammation-related microRNAs with microRNA-155 expression correlates with Th17 differentiation in patients with acute coronary syndrome
    Article Snippet: Total RNA was extracted from both freshly isolated PBMCs and plasma by TRIzol and TRIzol LS (Invitrogen, Carlsbad, CA, USA), respectively, according to the manufacturer's protocols, respectively. .. The concentration and purity of the RNA samples were measured by a spectrophotometer, and samples were used only if the ratio of the absorbance at 260 and 280 nm ( A 260/280 ) was between 1.8 and 2.0.

    Lysis:

    Article Title: Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix
    Article Snippet: The use of different RNA extraction kits – may affect the miRNA recovery from serum dry-spotted on pre-treated FTA Elute cards. .. To address this, we compared the performance of QIAzol lysis reagent with Trizol-LS reagent, Trizol reagent and column-based RNA isolation kit (Ambion). .. Our results showed that the average percentage miRNA recovery of all 10 miRNAs across these methods was similar (Supplementary Fig. ).

    Article Title: Circulating and tissue biomarkers in early-stage non-small cell lung cancer
    Article Snippet: Serum miRNA purification and expression profiling were performed according to Montani et al [ ]. .. Briefly, total RNA purification, including miRNAs, was based on lysis with guanidinium thiocyanate-phenol-chloroform extraction (TriZol-LS, Applied Biosystem) and Spin Column-based total RNA purification (MiRneasy Mini Kit, Qiagen). .. MiRNA qRT-PCR was carried out on a ViiA™ 7 instruments (ThermoFisher) using the manufacturer’s recommended cycling conditions.

    RNA Detection:

    Article Title: Laboratory Surveillance of Rabies in Humans, Domestic Animals, and Bats in Madagascar from 2005 to 2010
    Article Snippet: Paragraph title: 2.3. RABV RNA Detection ... RNA was extracted also from pools of bats blood clots supernatants or bats pharyngeal swabs VTM using TRIzol LS (Invitrogen, Carlsbad, Calif, USA) and from brain biopsies using TRIzol (Invitrogen, Carlsbad, Calif, USA), as recommended by the manufacturer.

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    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, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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