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  • 99
    Thermo Fisher trizol reagent
    Effects of aging on mitochondrial content, biogenesis, and structure in p66 Shc(−/−) mouse brain. (a) Quantification of <t>mtDNA</t> and nuclear DNA by qPCR. (b) The mtDNA/nDNA ratio was calculated from 3- and 24-month WT (grey) and p66 Shc(−/−) (black) groups. The mRNA levels of PGC-1 α were measured in <t>TRIzol-treated</t> brain extracts from all the studied groups; GADPH mRNA was used as standard ( n = 6). (c) Mitochondrial morphology was evaluated using electron microscopy of fixed brain slices ( n = 5 for each experimental group) (magnification:×20,000). (d) At least 300 tubular and fragmented mitochondria were counted per arbitrary area. The percentage distribution of tubular and fragmented brain mitochondria was determined in a minimum of 8–10 random fields at ×4400 magnification to ensure a representative area of analysis ( n = 5). Mitochondria whose length was more than three times their width were considered tubular, while the remaining round mitochondria were considered fragmented. The analysis was performed by two different investigators in a blinded fashion. Values represent the mean ± SEM; A represents p
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    Millipore trizol reagent
    E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total <t>RNA</t> was extracted using the <t>TRIzol</t> reagent, converted to cDNA using SuperScript VILO, and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value
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    99
    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 12258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher trizol plus rna purification kit
    Effect of knockout of BTN1A1 on lipogenic mRNA abundance in bovine mammary epithelial cells (BMEC) ( n = 3). Data are reported as Means ± SE. Total <t>RNA</t> was isolated using <t>TRIzol</t> reagent and the relative abundance of target genes was measured using real-time PCR.WT, wild-type; BTN1A1 (−/−) , BTN1A1 gene knockout cell (* P
    Trizol Plus Rna Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3455 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co trizol reagent
    Sirtuin 6 (SIRT6) silencing restricted hepatitis B virus (HBV) transcription and replication. (A – G) HepG2.2.15 and HBV-infected HepG2-sodium taurocholate cotransporting polypeptide (NTCP) cells were transfected with plasmids expressing short hairpin ribonucleic acids (shRNAs) targeting SIRT6 (shSIRT6-1 and shSIRT6-2) or scramble control shRNA (shCont). (A) Total protein was extracted at 4 days post-transfection and subjected to western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. Band intensities were quantified by ImageJ software and normalized to GAPDH. (B , C) HBV core deoxyribonucleic acid (DNA) were extracted at 5 days post-transfection. Then real-time polymerase chain reaction (PCR) and southern blotting were performed to detect HBV core DNA level. (D , E) After 4 days post transfection, total <t>RNA</t> was extracted by using <t>TRIzol</t> reagent and total HBV RNAs and 3.5-Kb RNA levels were detected by real-time PCR with specific primers. β-actin was used as the internal control. (F) The core protein was detected by immunofluorescence staining with indicated antibody at 4 days post-transfection and the images were collected by using confocal microscope. (G) HBV-infected HepG2-NTCP cells were transfected with plasmids expressing shRNAs targeting SIRT6 (shSIRT6-1 and shSIRT6-2) or scramble control shRNA (shCont). HBV covalently closed circular DNA (cccDNA) was extracted and applied for real time PCR. The ratios of total HBV RNAs/cccDNA and 3.5-Kb RNA/cccDNA were calculated. Data represented the mean ± SD of three independent experiments. *:P
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    Molecular Research Center inc trizol reagent
    Sirtuin 6 (SIRT6) silencing restricted hepatitis B virus (HBV) transcription and replication. (A – G) HepG2.2.15 and HBV-infected HepG2-sodium taurocholate cotransporting polypeptide (NTCP) cells were transfected with plasmids expressing short hairpin ribonucleic acids (shRNAs) targeting SIRT6 (shSIRT6-1 and shSIRT6-2) or scramble control shRNA (shCont). (A) Total protein was extracted at 4 days post-transfection and subjected to western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. Band intensities were quantified by ImageJ software and normalized to GAPDH. (B , C) HBV core deoxyribonucleic acid (DNA) were extracted at 5 days post-transfection. Then real-time polymerase chain reaction (PCR) and southern blotting were performed to detect HBV core DNA level. (D , E) After 4 days post transfection, total <t>RNA</t> was extracted by using <t>TRIzol</t> reagent and total HBV RNAs and 3.5-Kb RNA levels were detected by real-time PCR with specific primers. β-actin was used as the internal control. (F) The core protein was detected by immunofluorescence staining with indicated antibody at 4 days post-transfection and the images were collected by using confocal microscope. (G) HBV-infected HepG2-NTCP cells were transfected with plasmids expressing shRNAs targeting SIRT6 (shSIRT6-1 and shSIRT6-2) or scramble control shRNA (shCont). HBV covalently closed circular DNA (cccDNA) was extracted and applied for real time PCR. The ratios of total HBV RNAs/cccDNA and 3.5-Kb RNA/cccDNA were calculated. Data represented the mean ± SD of three independent experiments. *:P
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    Image Search Results


    Effects of aging on mitochondrial content, biogenesis, and structure in p66 Shc(−/−) mouse brain. (a) Quantification of mtDNA and nuclear DNA by qPCR. (b) The mtDNA/nDNA ratio was calculated from 3- and 24-month WT (grey) and p66 Shc(−/−) (black) groups. The mRNA levels of PGC-1 α were measured in TRIzol-treated brain extracts from all the studied groups; GADPH mRNA was used as standard ( n = 6). (c) Mitochondrial morphology was evaluated using electron microscopy of fixed brain slices ( n = 5 for each experimental group) (magnification:×20,000). (d) At least 300 tubular and fragmented mitochondria were counted per arbitrary area. The percentage distribution of tubular and fragmented brain mitochondria was determined in a minimum of 8–10 random fields at ×4400 magnification to ensure a representative area of analysis ( n = 5). Mitochondria whose length was more than three times their width were considered tubular, while the remaining round mitochondria were considered fragmented. The analysis was performed by two different investigators in a blinded fashion. Values represent the mean ± SEM; A represents p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: p66Shc Inactivation Modifies RNS Production, Regulates Sirt3 Activity, and Improves Mitochondrial Homeostasis, Delaying the Aging Process in Mouse Brain

    doi: 10.1155/2018/8561892

    Figure Lengend Snippet: Effects of aging on mitochondrial content, biogenesis, and structure in p66 Shc(−/−) mouse brain. (a) Quantification of mtDNA and nuclear DNA by qPCR. (b) The mtDNA/nDNA ratio was calculated from 3- and 24-month WT (grey) and p66 Shc(−/−) (black) groups. The mRNA levels of PGC-1 α were measured in TRIzol-treated brain extracts from all the studied groups; GADPH mRNA was used as standard ( n = 6). (c) Mitochondrial morphology was evaluated using electron microscopy of fixed brain slices ( n = 5 for each experimental group) (magnification:×20,000). (d) At least 300 tubular and fragmented mitochondria were counted per arbitrary area. The percentage distribution of tubular and fragmented brain mitochondria was determined in a minimum of 8–10 random fields at ×4400 magnification to ensure a representative area of analysis ( n = 5). Mitochondria whose length was more than three times their width were considered tubular, while the remaining round mitochondria were considered fragmented. The analysis was performed by two different investigators in a blinded fashion. Values represent the mean ± SEM; A represents p

    Article Snippet: RNA Extraction, Quantitative Real-Time PCR, and mtDNA Content Total RNA was extracted with TRIzol Reagent (Invitrogen Corp.).

    Techniques: Real-time Polymerase Chain Reaction, Pyrolysis Gas Chromatography, Electron Microscopy

    Time course of ozone-induced IL-8 mRNA expression in NHBE cells in the presence or absence of A 2 -isoprostane. NHBE cells cultured at ALI were exposed to FA or OZ over time (0.4 ppm; 0.5–5 h), and total RNA was isolated using TRIzol reagent. IL-8

    Journal: The Journal of Biological Chemistry

    Article Title: NADPH:Quinone Oxidoreductase 1 Regulates Host Susceptibility to Ozone via Isoprostane Generation *

    doi: 10.1074/jbc.M112.438440

    Figure Lengend Snippet: Time course of ozone-induced IL-8 mRNA expression in NHBE cells in the presence or absence of A 2 -isoprostane. NHBE cells cultured at ALI were exposed to FA or OZ over time (0.4 ppm; 0.5–5 h), and total RNA was isolated using TRIzol reagent. IL-8

    Article Snippet: RNA was isolated from NHBE cells using TRIzol (Invitrogen) according to the manufacturer's instructions, and IL-8 gene expression was analyzed by quantitative real time RT-PCR on an SDS 7300 machine (Applied Biosystems) in a 25-μl reaction that contained 1× TaqMan gene expression assay (mixture of PCR primer set specific for IL-8 and a FAM dye-labeled TaqMan MGB probe) using universal amplification conditions.

    Techniques: Expressing, Cell Culture, Isolation

    Transcriptome analyses of E10.5 Magoh , Rbm8a , and Eif4a3 haploinsufficient cortices reveal common downstream pathways. (A) Diagrammatic overview of RNA sequencing analysis of E10.5 neocortices from indicated genotypes. (B) qPCR showing expression of Magoh , Rbm8a , and Eif4a3 in their respective mutant E10.5 cortices. (C) Heatmaps showing z-score transformed normalized expression for all affected transcripts with an FDR corrected p-value, q

    Journal: PLoS Genetics

    Article Title: Haploinsufficiency for Core Exon Junction Complex Components Disrupts Embryonic Neurogenesis and Causes p53-Mediated Microcephaly

    doi: 10.1371/journal.pgen.1006282

    Figure Lengend Snippet: Transcriptome analyses of E10.5 Magoh , Rbm8a , and Eif4a3 haploinsufficient cortices reveal common downstream pathways. (A) Diagrammatic overview of RNA sequencing analysis of E10.5 neocortices from indicated genotypes. (B) qPCR showing expression of Magoh , Rbm8a , and Eif4a3 in their respective mutant E10.5 cortices. (C) Heatmaps showing z-score transformed normalized expression for all affected transcripts with an FDR corrected p-value, q

    Article Snippet: For qPCRs, whole neocortices from E10.5 and dorsal neocortices of E11.5, and E12.5 and E14.5 embryos were collected from C57BL/6J (wild-type), Emx1- Cre, Emx1- Cre;Eif4a3 lox /+, Emx1- Cre;Rbm8a lox /+ , and Emx1- Cre;Magoh lox /+ embryos and RNA was extracted using Trizol reagent (Invitrogen) followed by the RNeasy kit (Qiagen). cDNA was prepared according to the iScript kit (Bio-Rad). qPCR was performed in triplicates using Taqman probes (Life Technologies): Rbm8a (Mm04214345_s1), Eif4a3 (Mm00836350_g1), Magoh (Mm00487546_m1), Ngn2 (Mm00437603_g1), Tbr2 (Mm01351984_m1), Dclk1 (Mm00444950_m1) and Gapdh (4352339E).

    Techniques: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Expressing, Mutagenesis, Transformation Assay

    Transcriptome analyses of E10.5 Magoh germline haploinsufficient brains identifies alterations in ribosome and p53 signaling pathways. (A) Diagrammatic overview of RNA sequencing analysis of E10.5 neocortices (dotted lines) from indicated genotypes. (B) Heatmaps showing z-score transformed normalized expression for control and Magoh Mos2/+ . Genes and samples were clustered using correlation distance with complete linkage. (C) Scatter plot of transcripts significantly upregulated (green dots) and downregulated (red dots) in E10.5 Magoh Mos2/+ cortices ( q

    Journal: PLoS Genetics

    Article Title: Haploinsufficiency for Core Exon Junction Complex Components Disrupts Embryonic Neurogenesis and Causes p53-Mediated Microcephaly

    doi: 10.1371/journal.pgen.1006282

    Figure Lengend Snippet: Transcriptome analyses of E10.5 Magoh germline haploinsufficient brains identifies alterations in ribosome and p53 signaling pathways. (A) Diagrammatic overview of RNA sequencing analysis of E10.5 neocortices (dotted lines) from indicated genotypes. (B) Heatmaps showing z-score transformed normalized expression for control and Magoh Mos2/+ . Genes and samples were clustered using correlation distance with complete linkage. (C) Scatter plot of transcripts significantly upregulated (green dots) and downregulated (red dots) in E10.5 Magoh Mos2/+ cortices ( q

    Article Snippet: For qPCRs, whole neocortices from E10.5 and dorsal neocortices of E11.5, and E12.5 and E14.5 embryos were collected from C57BL/6J (wild-type), Emx1- Cre, Emx1- Cre;Eif4a3 lox /+, Emx1- Cre;Rbm8a lox /+ , and Emx1- Cre;Magoh lox /+ embryos and RNA was extracted using Trizol reagent (Invitrogen) followed by the RNeasy kit (Qiagen). cDNA was prepared according to the iScript kit (Bio-Rad). qPCR was performed in triplicates using Taqman probes (Life Technologies): Rbm8a (Mm04214345_s1), Eif4a3 (Mm00836350_g1), Magoh (Mm00487546_m1), Ngn2 (Mm00437603_g1), Tbr2 (Mm01351984_m1), Dclk1 (Mm00444950_m1) and Gapdh (4352339E).

    Techniques: RNA Sequencing Assay, Transformation Assay, Expressing

    Upregulation of CCL2/CCR2 is responsible for the IL-10-induced lung fibrocyte recruitment. A : BAL cells harvested from control and IL-10 OE mice were allowed to adhere for 2 h at 37°C and then, after removal of suspended cells, were subjected to total RNA preparation and Q-PCR analysis for CCR2, CCR4, and CXCR4 gene expression. B : whole lung total RNA was extracted by Trizol method and was subjected to Q-PCR analysis for CCL2, CCL19, CCL21b, and CXCL12 gene expression. C : CCL2 levels in the BAL fluids were measured by ELISA analysis. Results are ± SE of 4 animals and represent 2 independent experiments of a total number of 9–10 individual animals per group.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: New concepts of IL-10-induced lung fibrosis: fibrocyte recruitment and M2 activation in a CCL2/CCR2 axis

    doi: 10.1152/ajplung.00122.2010

    Figure Lengend Snippet: Upregulation of CCL2/CCR2 is responsible for the IL-10-induced lung fibrocyte recruitment. A : BAL cells harvested from control and IL-10 OE mice were allowed to adhere for 2 h at 37°C and then, after removal of suspended cells, were subjected to total RNA preparation and Q-PCR analysis for CCR2, CCR4, and CXCR4 gene expression. B : whole lung total RNA was extracted by Trizol method and was subjected to Q-PCR analysis for CCL2, CCL19, CCL21b, and CXCL12 gene expression. C : CCL2 levels in the BAL fluids were measured by ELISA analysis. Results are ± SE of 4 animals and represent 2 independent experiments of a total number of 9–10 individual animals per group.

    Article Snippet: Total RNA was isolated from cultured BAL cells or whole lung tissue with the Trizol method (Invitrogen Life Technologies) according to the manufacturer's protocol.

    Techniques: Mouse Assay, Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

    Agarose gel electrophoresis (1.00%). A) Showing RNA extracted using the modified TRIzol method; B) Showing RNA extracted using RNeasy Mini Kit. M: Molecular weight marker; COC: Cumulus oocyte complexes; Oo: Cumulus free oocytes; Oocyt: Cumulus free oocytes; stom: Stomach. C) Amplification of the control gene and beta-actin using RNA extracted through the modified TRIzol method. M: Molecular weight marker; B1, B2, and B3: Beta-actin gene; H3R: Histamine H3 receptor; H4R: Histamine H4 receptor; NC: Negative control. D) Amplification of the control gene, Gapdh using RNA extracted through RNeasy Mini Kit. M: Molecular weight marker; a, b and c: Gapdh; N: Negative control

    Journal: Veterinary Research Forum

    Article Title: The challenge of getting a high quality of RNA from oocyte for gene expression study

    doi: 10.30466/vrf.2018.92133.2230

    Figure Lengend Snippet: Agarose gel electrophoresis (1.00%). A) Showing RNA extracted using the modified TRIzol method; B) Showing RNA extracted using RNeasy Mini Kit. M: Molecular weight marker; COC: Cumulus oocyte complexes; Oo: Cumulus free oocytes; Oocyt: Cumulus free oocytes; stom: Stomach. C) Amplification of the control gene and beta-actin using RNA extracted through the modified TRIzol method. M: Molecular weight marker; B1, B2, and B3: Beta-actin gene; H3R: Histamine H3 receptor; H4R: Histamine H4 receptor; NC: Negative control. D) Amplification of the control gene, Gapdh using RNA extracted through RNeasy Mini Kit. M: Molecular weight marker; a, b and c: Gapdh; N: Negative control

    Article Snippet: In this study, the authors used‎ the RNA extracted from 90-100 mouse pre-implantation embryos and unfertilized oocytes using TRIzol reagents (Invitrogen Life Technologies, Karlsruhe, Germany) method.

    Techniques: Agarose Gel Electrophoresis, Modification, Molecular Weight, Marker, Amplification, Negative Control

    Analysis of SIV gag expression from the four different Kunjin SIV gag constructs. (A) Analysis of SIV Gag protein expression after infection of Vero cells with Kunjin VLPs. Cells were lysed after 48 h incubation and total cell lysates analysed by western blot using anti-Gag antibody (55-2F12). (B) As for A except that BHK cells were transfected with RNA for each of the vaccine constructs. (C) Comparison of SIV Gag and HIV Gag protein expression after infection of Vero cells as above with WT and HIV-1 VLPs using the AG3.0 antibody. (D) Comparison of the reactivity of AG3.0 in western analysis with serial dilutions of purified recombinant SIV Gag and recombinant HIV Gag proteins.

    Journal: Vaccine

    Article Title: Kunjin replicon-based simian immunodeficiency virus gag vaccines

    doi: 10.1016/j.vaccine.2008.04.001

    Figure Lengend Snippet: Analysis of SIV gag expression from the four different Kunjin SIV gag constructs. (A) Analysis of SIV Gag protein expression after infection of Vero cells with Kunjin VLPs. Cells were lysed after 48 h incubation and total cell lysates analysed by western blot using anti-Gag antibody (55-2F12). (B) As for A except that BHK cells were transfected with RNA for each of the vaccine constructs. (C) Comparison of SIV Gag and HIV Gag protein expression after infection of Vero cells as above with WT and HIV-1 VLPs using the AG3.0 antibody. (D) Comparison of the reactivity of AG3.0 in western analysis with serial dilutions of purified recombinant SIV Gag and recombinant HIV Gag proteins.

    Article Snippet: 2.4 Deletion mutant analyses Vero cells were transfected with Kunjin SIV gag VLPs at MOI = 1 and were cultured for 72 h. Total RNA was collected using Trizol reagent (Invitrogen) and SuperScript III reverse transcriptase (Invitrogen) was used to produce cDNA.

    Techniques: Expressing, Construct, Infection, Incubation, Western Blot, Transfection, Purification, Recombinant

    Analysis of insert deletions in the Kunjin SIV gag vaccines. (A) Non-denaturing agarose gel and ethidium bromide staining of in vitro transcribed RNA for the indicated constructs prior to transfection of packaging cells and VLP manufacture. Note the double-stranded DNA markers do not accurately illustrate the size of the transcribed single-stranded RNA species, which are about 10.5 kb. (B) Vero cells were infected with WT (lane 2), DX (lane 3), empty VLPs (lane 4) or nothing (lane 5) and RT-PCR performed on extracted RNA using primers flanking the multiple cloning site region into which the gag genes were inserted for the former two constructs. Lane 1 shows the markers and lane 6 no template control. (C) Sequencing of the ≈180 bp fragment from DX transfected cells. The insert deletion is schematically represented with the sequence either side of the insert deletion site given beneath.

    Journal: Vaccine

    Article Title: Kunjin replicon-based simian immunodeficiency virus gag vaccines

    doi: 10.1016/j.vaccine.2008.04.001

    Figure Lengend Snippet: Analysis of insert deletions in the Kunjin SIV gag vaccines. (A) Non-denaturing agarose gel and ethidium bromide staining of in vitro transcribed RNA for the indicated constructs prior to transfection of packaging cells and VLP manufacture. Note the double-stranded DNA markers do not accurately illustrate the size of the transcribed single-stranded RNA species, which are about 10.5 kb. (B) Vero cells were infected with WT (lane 2), DX (lane 3), empty VLPs (lane 4) or nothing (lane 5) and RT-PCR performed on extracted RNA using primers flanking the multiple cloning site region into which the gag genes were inserted for the former two constructs. Lane 1 shows the markers and lane 6 no template control. (C) Sequencing of the ≈180 bp fragment from DX transfected cells. The insert deletion is schematically represented with the sequence either side of the insert deletion site given beneath.

    Article Snippet: 2.4 Deletion mutant analyses Vero cells were transfected with Kunjin SIV gag VLPs at MOI = 1 and were cultured for 72 h. Total RNA was collected using Trizol reagent (Invitrogen) and SuperScript III reverse transcriptase (Invitrogen) was used to produce cDNA.

    Techniques: Agarose Gel Electrophoresis, Staining, In Vitro, Construct, Transfection, Infection, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Sequencing

    Regulation of epithelial and mesenchymal markers by IL-17A, TNF-α, IL-8, TGF-β1 and IL-13 at the RNA level. Psoriatic epidermal keratinocytes were starved for 24 h and then incubated with 50 ng ml −1 IL-17A, TNF-α, IL-8, TGF-β1 and IL-13 for 24 h. Then total RNA was extracted by the TRIZol kit. ( a ) The expression of target genes is indicated as RPKM determined by RNAseq and ( b ) the ratio of the target gene to β-actin determined by qRT-PCR.

    Journal: Open Biology

    Article Title: Analysis of epithelial–mesenchymal transition markers in psoriatic epidermal keratinocytes

    doi: 10.1098/rsob.150032

    Figure Lengend Snippet: Regulation of epithelial and mesenchymal markers by IL-17A, TNF-α, IL-8, TGF-β1 and IL-13 at the RNA level. Psoriatic epidermal keratinocytes were starved for 24 h and then incubated with 50 ng ml −1 IL-17A, TNF-α, IL-8, TGF-β1 and IL-13 for 24 h. Then total RNA was extracted by the TRIZol kit. ( a ) The expression of target genes is indicated as RPKM determined by RNAseq and ( b ) the ratio of the target gene to β-actin determined by qRT-PCR.

    Article Snippet: RNA sequencing RNA was extracted from normal (n = 3) and psoriatic (n = 3) keratinocytes cultured in vitro by using TRIZol kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

    Techniques: Incubation, Expressing, Quantitative RT-PCR

    Expression of SPARC in D283 medulloblastoma cells Medulloblastoma cells were transfected with plasmid containing full-length SPARC cDNA (pSPARC) or empty vehicle control (pEV) or mock (untreated) control for 36 hrs. (A) SPARC protein levels were determined in total cell lysates by western blotting analysis using SPARC specific primary antibody. GAPDH was used to confirm equal loading. (B) Total RNA was extracted using Trizol reagent, and qRT-PCR was performed for SPARC mRNA transcript level. (C) p53 protein levels were determined in total cell lysates by western blotting analysis using p53 specific primary antibody. GAPDH was used to confirm equal loading. (D) Total RNA was extracted using Trizol reagent, and qRT-PCR was performed for p53 mRNA transcript level or (E) p21 mRNA transcript level. Total protein levels were quantified by densitometric analysis as shown in the corresponding bar graph. ** P

    Journal: Genes & Cancer

    Article Title: SPARC overexpression alters microRNA expression profiles involved in tumor progression

    doi: 10.18632/genesandcancer.130

    Figure Lengend Snippet: Expression of SPARC in D283 medulloblastoma cells Medulloblastoma cells were transfected with plasmid containing full-length SPARC cDNA (pSPARC) or empty vehicle control (pEV) or mock (untreated) control for 36 hrs. (A) SPARC protein levels were determined in total cell lysates by western blotting analysis using SPARC specific primary antibody. GAPDH was used to confirm equal loading. (B) Total RNA was extracted using Trizol reagent, and qRT-PCR was performed for SPARC mRNA transcript level. (C) p53 protein levels were determined in total cell lysates by western blotting analysis using p53 specific primary antibody. GAPDH was used to confirm equal loading. (D) Total RNA was extracted using Trizol reagent, and qRT-PCR was performed for p53 mRNA transcript level or (E) p21 mRNA transcript level. Total protein levels were quantified by densitometric analysis as shown in the corresponding bar graph. ** P

    Article Snippet: Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from D283 mock or pEV control and pSPARC cells using TRIzol® solution (Invitrogen, Carlsbad, CA) as per standard protocol.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR

    Cytokine gene expression is reduced during CHIKV Opal524R infection. (A, B) Twenty-four-day-old C57BL6/J mice were infected in the left rear footpad with 100 PFU of wild-type CHIKV ( n = 5) or Opal524R ( n = 5) or mock infected with diluent ( n = 3). At days 1 (A) and 2 (B) postinfection, mice were sacrificed and perfused intracardially with 1× PBS, and the ipsilateral foot was harvested in TRIzol. RNA was extracted, cDNA was generated, and cytokine gene expression was assessed by quantitative real-time PCR. Each cytokine was normalized to host 18S gene expression. Fold changes in gene expression relative to mock infection (left) and wild-type infection (right) are shown. Data are representative of two independent experiments. Statistical significance is indicated as follows: *, P

    Journal: mBio

    Article Title: Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology

    doi: 10.1128/mBio.01456-17

    Figure Lengend Snippet: Cytokine gene expression is reduced during CHIKV Opal524R infection. (A, B) Twenty-four-day-old C57BL6/J mice were infected in the left rear footpad with 100 PFU of wild-type CHIKV ( n = 5) or Opal524R ( n = 5) or mock infected with diluent ( n = 3). At days 1 (A) and 2 (B) postinfection, mice were sacrificed and perfused intracardially with 1× PBS, and the ipsilateral foot was harvested in TRIzol. RNA was extracted, cDNA was generated, and cytokine gene expression was assessed by quantitative real-time PCR. Each cytokine was normalized to host 18S gene expression. Fold changes in gene expression relative to mock infection (left) and wild-type infection (right) are shown. Data are representative of two independent experiments. Statistical significance is indicated as follows: *, P

    Article Snippet: RNA was isolated from purified virus preparations by TRIzol extraction (Ambion).

    Techniques: Expressing, Infection, Mouse Assay, Generated, Real-time Polymerase Chain Reaction

    Survivin expression and oncolytic adenovirus replicative activity in cell lines examined (A) Cultured gastric cancer and normal cells were collected, and the TRIzol method was used to extract the total RNA from these cells. Survivin was amplified from the extracted RNA through RT-PCR with GAPDH as the internal control. (B) Fluorescence microscopy was used to observe EGFP-positive gastric cancer cells and normal cells 48 h after the cells were infected with AdSurp-EGFP or AdCMV-EGFP at an MOI of 1 pfu/cell, original magnification 200×. (C) Gastric cancer and normal cells were infected with AdSurp-Hsp70 or AdCMV-Hsp70 at an MOI of 1 pfu/cell. Forty-eight hours after infection, the cells were collected and the virus titer was quantified using the TCID 50 assay. ** P

    Journal: Oncotarget

    Article Title: Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy

    doi:

    Figure Lengend Snippet: Survivin expression and oncolytic adenovirus replicative activity in cell lines examined (A) Cultured gastric cancer and normal cells were collected, and the TRIzol method was used to extract the total RNA from these cells. Survivin was amplified from the extracted RNA through RT-PCR with GAPDH as the internal control. (B) Fluorescence microscopy was used to observe EGFP-positive gastric cancer cells and normal cells 48 h after the cells were infected with AdSurp-EGFP or AdCMV-EGFP at an MOI of 1 pfu/cell, original magnification 200×. (C) Gastric cancer and normal cells were infected with AdSurp-Hsp70 or AdCMV-Hsp70 at an MOI of 1 pfu/cell. Forty-eight hours after infection, the cells were collected and the virus titer was quantified using the TCID 50 assay. ** P

    Article Snippet: The total RNA was extracted from the cells using the TRIzol reagent kit (Invitrogen Life Technologies, Carlsbad, CA, USA) in accordance with the manufacturer's instructions.

    Techniques: Expressing, Activity Assay, Cell Culture, Amplification, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Microscopy, Infection

    Simultaneous optimization of EDTA, NaCl, and SDS concentrations enhances protein yield during solubilization of TRIzol-precipitated protein

    Journal: Journal of neuroscience methods

    Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue

    doi: 10.1016/j.jneumeth.2017.02.002

    Figure Lengend Snippet: Simultaneous optimization of EDTA, NaCl, and SDS concentrations enhances protein yield during solubilization of TRIzol-precipitated protein

    Article Snippet: In fact, the TRIzol protocol (Life Technologies) recommends solubilization in 1% SDS, and indicates that dialysis and subsequent reconstitution in an SDS-urea solution may improve yield (Life Technologies; November 9, 2016 edition).

    Techniques:

    3.1 EDTA, NaCl, and SDS concentrations modulate protein yield during solubilization of TRIzol-precipitated protein

    Journal: Journal of neuroscience methods

    Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue

    doi: 10.1016/j.jneumeth.2017.02.002

    Figure Lengend Snippet: 3.1 EDTA, NaCl, and SDS concentrations modulate protein yield during solubilization of TRIzol-precipitated protein

    Article Snippet: In fact, the TRIzol protocol (Life Technologies) recommends solubilization in 1% SDS, and indicates that dialysis and subsequent reconstitution in an SDS-urea solution may improve yield (Life Technologies; November 9, 2016 edition).

    Techniques:

    EDTA, NaCl, and SDS concentrations significantly alter protein yield during solubilization of TRIzol-precipitated protein

    Journal: Journal of neuroscience methods

    Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue

    doi: 10.1016/j.jneumeth.2017.02.002

    Figure Lengend Snippet: EDTA, NaCl, and SDS concentrations significantly alter protein yield during solubilization of TRIzol-precipitated protein

    Article Snippet: In fact, the TRIzol protocol (Life Technologies) recommends solubilization in 1% SDS, and indicates that dialysis and subsequent reconstitution in an SDS-urea solution may improve yield (Life Technologies; November 9, 2016 edition).

    Techniques:

    Laser-capture microdissection of glial cells and vessels. A: The target normal glial cell population was dissected with ultraviolet laser. B: The dissected cells were photo-ablated within the laser focus and then directly collected into a microcentrifuge tube cap filled with Trizol for RNA isolation by laser pressure catapulting. C: The target tumor cell population (glioblastoma) was dissected with ultraviolet laser. D: The dissected cells were photo-ablated and collected as mentioned under B . E: The targeted hyperplastic vessel in pilocytic astrocytoma was positioned. F: The dissected vessel was photo-ablated and collected as mentioned in ( B ).

    Journal: The American Journal of Pathology

    Article Title: Differential Expression of Splicing Variants of the Human Caldesmon Gene (CALD1) in Glioma Neovascularization versus Normal Brain Microvasculature

    doi:

    Figure Lengend Snippet: Laser-capture microdissection of glial cells and vessels. A: The target normal glial cell population was dissected with ultraviolet laser. B: The dissected cells were photo-ablated within the laser focus and then directly collected into a microcentrifuge tube cap filled with Trizol for RNA isolation by laser pressure catapulting. C: The target tumor cell population (glioblastoma) was dissected with ultraviolet laser. D: The dissected cells were photo-ablated and collected as mentioned under B . E: The targeted hyperplastic vessel in pilocytic astrocytoma was positioned. F: The dissected vessel was photo-ablated and collected as mentioned in ( B ).

    Article Snippet: Total cellular protein was co-isolated and lysed during RNA isolation by Trizol according to the manufacturer’s protocol (Invitrogen).

    Techniques: Laser Capture Microdissection, Isolation

    E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total RNA was extracted using the TRIzol reagent, converted to cDNA using SuperScript VILO, and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value

    Journal: Journal of Virology

    Article Title: Adenovirus 5 E1A Interacts with E4orf3 To Regulate Viral Chromatin Organization

    doi: 10.1128/JVI.00157-19

    Figure Lengend Snippet: E4orf3 inhibits viral gene expression and affects viral chromatin organization. (A) A549-E1A289R cells that stably express HAdV5 E1A289R were infected with either Ad-CMV, which expresses no E1A or E4orf3, or Ad-CMV-E4orf3, which expresses an HA-tagged E4orf3 protein. Sixteen hours after infection, total RNA was extracted using the TRIzol reagent, converted to cDNA using SuperScript VILO, and analyzed for expression using real-time qPCR with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was determined as percentage of the GAPDH mRNA level. Since E4orf3 was provided by the virus and Ad-CMV has no extra copy of the E4orf3 gene, we used E4orf6/7 to analyze the expression from the E4 transcriptional unit. Statistically significant differences ( P value ≤ 0.0123) between the two viruses are indicated with an asterisk. Error bars represent SDs from three biological replicates. (B) A549-E1A289R cells were infected at an MOI of 10 for 24 h. Cells were subsequently fixed with paraformaldehyde 24 h after infection, and chromatin was immunoprecipitated using an anti-E1A antibody cocktail consisting of an equal mix of M58 and M73 hybridoma supernatants or a rabbit anti-rat IgG control antibody. Promoter occupancy of E1A was assayed using real-time qPCR using ABI SuperMix for CFX with a Bio-Rad CFX96 real-time instrument. The inset shows total E1A immunoprecipitated from the samples. Results are represented as percentage of input. Statistically significant differences ( P value

    Article Snippet: Total RNA was extracted using the TRIzol reagent (Sigma) at the desired time points according to manufacturer’s instructions.

    Techniques: Expressing, Stable Transfection, Infection, Real-time Polymerase Chain Reaction, Immunoprecipitation

    E1A interaction with E4orf3 affect expression of viral early genes and influences viral chromatin structure. (A) IMR-90 cells were grown until confluence, at which point medium was replaced and the cells were allowed to grow for another 72 h. At that point cells were infected with either dl 309 or Ad5.Δ13-15 at an MOI of 30 for 1 h, after which the saved medium originally removed from the cells was reapplied. Cells were then incubated for the indicated time, after which RNA was extracted using the TRIzol reagent, converted to cDNA using SuperScript VILO, and analyzed for expression using real-time quantitative PCR (qPCR) with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was performed using the Pfaffl method, comparing the expression in Ad5.Δ13-15-infected cells to that in dl 309-infected cells. Expression of GAPDH was used to normalize the results. Statistically significant differences ( P value

    Journal: Journal of Virology

    Article Title: Adenovirus 5 E1A Interacts with E4orf3 To Regulate Viral Chromatin Organization

    doi: 10.1128/JVI.00157-19

    Figure Lengend Snippet: E1A interaction with E4orf3 affect expression of viral early genes and influences viral chromatin structure. (A) IMR-90 cells were grown until confluence, at which point medium was replaced and the cells were allowed to grow for another 72 h. At that point cells were infected with either dl 309 or Ad5.Δ13-15 at an MOI of 30 for 1 h, after which the saved medium originally removed from the cells was reapplied. Cells were then incubated for the indicated time, after which RNA was extracted using the TRIzol reagent, converted to cDNA using SuperScript VILO, and analyzed for expression using real-time quantitative PCR (qPCR) with the Bio-Rad CFX96 instrument and ABI SuperMix for CFX reagent. Analysis of expression was performed using the Pfaffl method, comparing the expression in Ad5.Δ13-15-infected cells to that in dl 309-infected cells. Expression of GAPDH was used to normalize the results. Statistically significant differences ( P value

    Article Snippet: Total RNA was extracted using the TRIzol reagent (Sigma) at the desired time points according to manufacturer’s instructions.

    Techniques: Expressing, Infection, Incubation, Real-time Polymerase Chain Reaction

    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

    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

    Effect of knockout of BTN1A1 on lipogenic mRNA abundance in bovine mammary epithelial cells (BMEC) ( n = 3). Data are reported as Means ± SE. Total RNA was isolated using TRIzol reagent and the relative abundance of target genes was measured using real-time PCR.WT, wild-type; BTN1A1 (−/−) , BTN1A1 gene knockout cell (* P

    Journal: Journal of Animal Science and Biotechnology

    Article Title: Knockout of butyrophilin subfamily 1 member A1 (BTN1A1) alters lipid droplet formation and phospholipid composition in bovine mammary epithelial cells

    doi: 10.1186/s40104-020-00479-6

    Figure Lengend Snippet: Effect of knockout of BTN1A1 on lipogenic mRNA abundance in bovine mammary epithelial cells (BMEC) ( n = 3). Data are reported as Means ± SE. Total RNA was isolated using TRIzol reagent and the relative abundance of target genes was measured using real-time PCR.WT, wild-type; BTN1A1 (−/−) , BTN1A1 gene knockout cell (* P

    Article Snippet: After culturing with lactogenic medium for 48 h, total RNA was extracted from BMEC using a TRIzol Plus RNA Purification Kit (12183555, Thermo Fisher Scientific) according to the manufacturer’s protocol.

    Techniques: Knock-Out, Isolation, Real-time Polymerase Chain Reaction, Gene Knockout

    Sirtuin 6 (SIRT6) silencing restricted hepatitis B virus (HBV) transcription and replication. (A – G) HepG2.2.15 and HBV-infected HepG2-sodium taurocholate cotransporting polypeptide (NTCP) cells were transfected with plasmids expressing short hairpin ribonucleic acids (shRNAs) targeting SIRT6 (shSIRT6-1 and shSIRT6-2) or scramble control shRNA (shCont). (A) Total protein was extracted at 4 days post-transfection and subjected to western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. Band intensities were quantified by ImageJ software and normalized to GAPDH. (B , C) HBV core deoxyribonucleic acid (DNA) were extracted at 5 days post-transfection. Then real-time polymerase chain reaction (PCR) and southern blotting were performed to detect HBV core DNA level. (D , E) After 4 days post transfection, total RNA was extracted by using TRIzol reagent and total HBV RNAs and 3.5-Kb RNA levels were detected by real-time PCR with specific primers. β-actin was used as the internal control. (F) The core protein was detected by immunofluorescence staining with indicated antibody at 4 days post-transfection and the images were collected by using confocal microscope. (G) HBV-infected HepG2-NTCP cells were transfected with plasmids expressing shRNAs targeting SIRT6 (shSIRT6-1 and shSIRT6-2) or scramble control shRNA (shCont). HBV covalently closed circular DNA (cccDNA) was extracted and applied for real time PCR. The ratios of total HBV RNAs/cccDNA and 3.5-Kb RNA/cccDNA were calculated. Data represented the mean ± SD of three independent experiments. *:P

    Journal: Frontiers in Pharmacology

    Article Title: SIRT6 Inhibitor, OSS_128167 Restricts Hepatitis B Virus Transcription and Replication Through Targeting Transcription Factor Peroxisome Proliferator-Activated Receptors α

    doi: 10.3389/fphar.2019.01270

    Figure Lengend Snippet: Sirtuin 6 (SIRT6) silencing restricted hepatitis B virus (HBV) transcription and replication. (A – G) HepG2.2.15 and HBV-infected HepG2-sodium taurocholate cotransporting polypeptide (NTCP) cells were transfected with plasmids expressing short hairpin ribonucleic acids (shRNAs) targeting SIRT6 (shSIRT6-1 and shSIRT6-2) or scramble control shRNA (shCont). (A) Total protein was extracted at 4 days post-transfection and subjected to western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. Band intensities were quantified by ImageJ software and normalized to GAPDH. (B , C) HBV core deoxyribonucleic acid (DNA) were extracted at 5 days post-transfection. Then real-time polymerase chain reaction (PCR) and southern blotting were performed to detect HBV core DNA level. (D , E) After 4 days post transfection, total RNA was extracted by using TRIzol reagent and total HBV RNAs and 3.5-Kb RNA levels were detected by real-time PCR with specific primers. β-actin was used as the internal control. (F) The core protein was detected by immunofluorescence staining with indicated antibody at 4 days post-transfection and the images were collected by using confocal microscope. (G) HBV-infected HepG2-NTCP cells were transfected with plasmids expressing shRNAs targeting SIRT6 (shSIRT6-1 and shSIRT6-2) or scramble control shRNA (shCont). HBV covalently closed circular DNA (cccDNA) was extracted and applied for real time PCR. The ratios of total HBV RNAs/cccDNA and 3.5-Kb RNA/cccDNA were calculated. Data represented the mean ± SD of three independent experiments. *:P

    Article Snippet: Ribonucleic Acid Extraction and Reverse Transcription The RNA was extracted with TRIzol reagent (TianGen), and precipitated with isopropyl alcohol.

    Techniques: Infection, Transfection, Expressing, shRNA, Western Blot, Software, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Southern Blot, Immunofluorescence, Staining, Microscopy

    Sirtuin 6 (SIRT6) enhanced the activity of hepatitis B virus (HBV) core promoter through upregulating transcription factor peroxisome proliferator-activated receptors α (PPARα). (A) Four luciferase reporter constructs were cotransfected with Flag-SIRT6 into Huh7 cells, and the renilla luciferase reporter (RL-TK) was cotransfected to normalized transfection efficiency. The luciferase activities were determined 2 days after transfection. (B) HepG2.2.15 cells were transfected with plasmids expressing Flag-SIRT6. After 3 days post-transfection, total ribonucleic acid (RNA) was extracted by using TRIzol reagent and the expression of various transcription factors associated to HBV transcription were detected by real-time polymerase chain reaction (PCR) with specific primers. β-actin was used as the internal control. (C) HepG2.2.15 cells were transfected with plasmids expressing Flag-SIRT6. Total protein was extracted at 3 days post-transfection and subjected to western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. (D) HepG2.2.15 cells were transfected with plasmids expressing shRNAs targeting SIRT6 (shSIRT6-1 and shSIRT6-2) or scramble control short hairpin RNA (shRNA) (shCont). After 3 days post-transfection, total RNA was extracted by using TRIzol reagent and the expression of PPARα was detected by real-time PCR with specific primers. β-actin was used as the internal control. At the same time, total protein was extracted and subjected to western blotting. GAPDH was used as the loading control. Band intensities were quantified by ImageJ software and normalized to GAPDH. (E) HepG2.2.15 cells were treated with OSS_128167 for 3 days. The expression of PPARα was detected by real-time PCR with specific primers. β-Actin was used as the internal control. And total protein was subjected to western blotting. GAPDH was used as the loading control. Band intensities were quantified by ImageJ software and normalized to GAPDH. (F) The Huh7 cells were transfected with pGL3-Cp and RL-TK 24 h after transfection of plasmids expressing PPARα shRNA or scramble control shRNA. The luciferase activity was determined at 72 h posttransfection. RL-TK was used to normalized transfection efficiency. (G – J) HBV-infected HepG2-sodium taurocholate cotransporting polypeptide (NTCP) cells were transfected with plasmids expressing shRNAs targeting PPARα (shPPARα-1 and shPPARα-2) or scramble control shRNA (shCont). (G) HBV core deoxyribonucleic acid (DNA) were extracted at 5 days post-transfection. Then real-time PCR was performed to detect HBV core DNA level. (H) After 4 days post-transfection, total RNA was extracted by using TRIzol reagent and 3.5-Kb RNA level were detected by real-time PCR with specific primers. β-actin was used as the internal control. (I – J) Secretion of hepatitis B surface antigen (HBsAg) and hepatitis B envelope antigen (HBeAg) were assayed by using ELISA 4 days after transfection. At the same time, HBsAg production in cell lysates was determined by western blotting. GAPDH was used as the loading control. Data represented the mean ± SD of three independent experiments. *:P

    Journal: Frontiers in Pharmacology

    Article Title: SIRT6 Inhibitor, OSS_128167 Restricts Hepatitis B Virus Transcription and Replication Through Targeting Transcription Factor Peroxisome Proliferator-Activated Receptors α

    doi: 10.3389/fphar.2019.01270

    Figure Lengend Snippet: Sirtuin 6 (SIRT6) enhanced the activity of hepatitis B virus (HBV) core promoter through upregulating transcription factor peroxisome proliferator-activated receptors α (PPARα). (A) Four luciferase reporter constructs were cotransfected with Flag-SIRT6 into Huh7 cells, and the renilla luciferase reporter (RL-TK) was cotransfected to normalized transfection efficiency. The luciferase activities were determined 2 days after transfection. (B) HepG2.2.15 cells were transfected with plasmids expressing Flag-SIRT6. After 3 days post-transfection, total ribonucleic acid (RNA) was extracted by using TRIzol reagent and the expression of various transcription factors associated to HBV transcription were detected by real-time polymerase chain reaction (PCR) with specific primers. β-actin was used as the internal control. (C) HepG2.2.15 cells were transfected with plasmids expressing Flag-SIRT6. Total protein was extracted at 3 days post-transfection and subjected to western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. (D) HepG2.2.15 cells were transfected with plasmids expressing shRNAs targeting SIRT6 (shSIRT6-1 and shSIRT6-2) or scramble control short hairpin RNA (shRNA) (shCont). After 3 days post-transfection, total RNA was extracted by using TRIzol reagent and the expression of PPARα was detected by real-time PCR with specific primers. β-actin was used as the internal control. At the same time, total protein was extracted and subjected to western blotting. GAPDH was used as the loading control. Band intensities were quantified by ImageJ software and normalized to GAPDH. (E) HepG2.2.15 cells were treated with OSS_128167 for 3 days. The expression of PPARα was detected by real-time PCR with specific primers. β-Actin was used as the internal control. And total protein was subjected to western blotting. GAPDH was used as the loading control. Band intensities were quantified by ImageJ software and normalized to GAPDH. (F) The Huh7 cells were transfected with pGL3-Cp and RL-TK 24 h after transfection of plasmids expressing PPARα shRNA or scramble control shRNA. The luciferase activity was determined at 72 h posttransfection. RL-TK was used to normalized transfection efficiency. (G – J) HBV-infected HepG2-sodium taurocholate cotransporting polypeptide (NTCP) cells were transfected with plasmids expressing shRNAs targeting PPARα (shPPARα-1 and shPPARα-2) or scramble control shRNA (shCont). (G) HBV core deoxyribonucleic acid (DNA) were extracted at 5 days post-transfection. Then real-time PCR was performed to detect HBV core DNA level. (H) After 4 days post-transfection, total RNA was extracted by using TRIzol reagent and 3.5-Kb RNA level were detected by real-time PCR with specific primers. β-actin was used as the internal control. (I – J) Secretion of hepatitis B surface antigen (HBsAg) and hepatitis B envelope antigen (HBeAg) were assayed by using ELISA 4 days after transfection. At the same time, HBsAg production in cell lysates was determined by western blotting. GAPDH was used as the loading control. Data represented the mean ± SD of three independent experiments. *:P

    Article Snippet: Ribonucleic Acid Extraction and Reverse Transcription The RNA was extracted with TRIzol reagent (TianGen), and precipitated with isopropyl alcohol.

    Techniques: Activity Assay, Luciferase, Construct, Transfection, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Western Blot, shRNA, Software, Infection, Enzyme-linked Immunosorbent Assay

    Sirtuin 6 (SIRT6) overexpression promoted hepatitis B virus (HBV) transcription depends on its deacetylase activity. (A – E) HepG2.2.15 and HBV-infected HepG2-sodium taurocholate cotransporting polypeptide (NTCP) cells were transfected with plasmids expressing Flag-SIRT6 and exposed to OSS_128167 at 1 day post-transfection. (A – B) Total protein was extracted at 3 days post-transfection and subjected to western blotting. Glyceraldehyde 3-phosphate dehydrogenase and H3 were used as the loading control, respectively. Band intensities were quantified by ImageJ software and normalized to H3. (C) HBV core deoxyribonucleic acid (DNA) were extracted at 4 days post-transfection. Then real-time polymerase chain reaction (PCR) and southern blotting were performed to detect HBV core DNA level. (D) After 3 days post-transfection, total ribonucleic acid (RNA) was extracted by using TRIzol reagent and total HBV RNAs and 3.5-Kb RNA levels were detected by real-time PCR with specific primers. β-actin was used as the internal control. (E) HBV-infected HepG2-NTCP cells were transfected with plasmids expressing Flag-SIRT6, then treated with OSS_128167 1 day later. HBV covalently closed circular DNA (cccDNA) was extracted and applied for real time PCR. The ratios of total HBV RNAs/cccDNA and 3.5-Kb RNA/cccDNA were calculated. Data represented the mean ± SD of three independent experiments. *:P

    Journal: Frontiers in Pharmacology

    Article Title: SIRT6 Inhibitor, OSS_128167 Restricts Hepatitis B Virus Transcription and Replication Through Targeting Transcription Factor Peroxisome Proliferator-Activated Receptors α

    doi: 10.3389/fphar.2019.01270

    Figure Lengend Snippet: Sirtuin 6 (SIRT6) overexpression promoted hepatitis B virus (HBV) transcription depends on its deacetylase activity. (A – E) HepG2.2.15 and HBV-infected HepG2-sodium taurocholate cotransporting polypeptide (NTCP) cells were transfected with plasmids expressing Flag-SIRT6 and exposed to OSS_128167 at 1 day post-transfection. (A – B) Total protein was extracted at 3 days post-transfection and subjected to western blotting. Glyceraldehyde 3-phosphate dehydrogenase and H3 were used as the loading control, respectively. Band intensities were quantified by ImageJ software and normalized to H3. (C) HBV core deoxyribonucleic acid (DNA) were extracted at 4 days post-transfection. Then real-time polymerase chain reaction (PCR) and southern blotting were performed to detect HBV core DNA level. (D) After 3 days post-transfection, total ribonucleic acid (RNA) was extracted by using TRIzol reagent and total HBV RNAs and 3.5-Kb RNA levels were detected by real-time PCR with specific primers. β-actin was used as the internal control. (E) HBV-infected HepG2-NTCP cells were transfected with plasmids expressing Flag-SIRT6, then treated with OSS_128167 1 day later. HBV covalently closed circular DNA (cccDNA) was extracted and applied for real time PCR. The ratios of total HBV RNAs/cccDNA and 3.5-Kb RNA/cccDNA were calculated. Data represented the mean ± SD of three independent experiments. *:P

    Article Snippet: Ribonucleic Acid Extraction and Reverse Transcription The RNA was extracted with TRIzol reagent (TianGen), and precipitated with isopropyl alcohol.

    Techniques: Over Expression, Histone Deacetylase Assay, Activity Assay, Infection, Transfection, Expressing, Western Blot, Software, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Southern Blot