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  • 99
    Thermo Fisher deoxyribonucleic acid dna free kit
    Comparison of <t>RNA</t> snap ™ and Trizol® Max™ isolated RNA in an RT–PCR experiment. RNA isolated from SK4390 ( rph-1 ΔrppH ) was reversed transcribed with a primer specific for the lpp mRNA (∼330 nt) and subsequently PCR amplified for either 5,10,15, or 20 cycles (see ‘Materials and Methods’ section). The amplified PCR products were run on a 2% agarose gel and quantitated using ImageQuant TL software (GE). The amount of PCR product at the end of a fixed number of cycles from RNA snap ™ isolated RNA was set at 1 and compared with the amount of product obtained using Trizol® Max™ isolated RNA. Lanes 1 and 12, Gene Ruler™ Low Range <t>DNA</t> Ladder (Fermentas).
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    Thermo Fisher 0535s turbo dna free kit thermo fisher scientific cat
    Comparison of <t>RNA</t> snap ™ and Trizol® Max™ isolated RNA in an RT–PCR experiment. RNA isolated from SK4390 ( rph-1 ΔrppH ) was reversed transcribed with a primer specific for the lpp mRNA (∼330 nt) and subsequently PCR amplified for either 5,10,15, or 20 cycles (see ‘Materials and Methods’ section). The amplified PCR products were run on a 2% agarose gel and quantitated using ImageQuant TL software (GE). The amount of PCR product at the end of a fixed number of cycles from RNA snap ™ isolated RNA was set at 1 and compared with the amount of product obtained using Trizol® Max™ isolated RNA. Lanes 1 and 12, Gene Ruler™ Low Range <t>DNA</t> Ladder (Fermentas).
    0535s Turbo Dna Free Kit Thermo Fisher Scientific Cat, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Comparison of <t>RNA</t> snap ™ and Trizol® Max™ isolated RNA in an RT–PCR experiment. RNA isolated from SK4390 ( rph-1 ΔrppH ) was reversed transcribed with a primer specific for the lpp mRNA (∼330 nt) and subsequently PCR amplified for either 5,10,15, or 20 cycles (see ‘Materials and Methods’ section). The amplified PCR products were run on a 2% agarose gel and quantitated using ImageQuant TL software (GE). The amount of PCR product at the end of a fixed number of cycles from RNA snap ™ isolated RNA was set at 1 and compared with the amount of product obtained using Trizol® Max™ isolated RNA. Lanes 1 and 12, Gene Ruler™ Low Range <t>DNA</t> Ladder (Fermentas).
    Turbo Deoxyribonucleic Acid Dna Free Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    INS sequence chromatographs for imprint analysis in the pouch young liver. Direct sequence analysis for INS in the pouch young liver. Chromatogram traces of genomic <t>DNA</t> (gDNA) from the mother and pouch young and of cDNA from the pouch young liver. ( A ) The pouch young inherited allele 2 (G-T) from its mother, and the clear monoallelic expression of allele 3 (A-T) in the liver was inherited from the father. ( B ) The pouch young inherited allele 1 (G-C) from its mother, and the clear monoallelic expression of allele 2 (G-T) in the liver was inherited from the father. <t>RNA</t> was extracted twice from the same liver sample and direct sequencing produced the same results in both samples. These results indicate that INS expression in the liver is a result of parent-of-origin specific genomic imprinting and not random monoallelic expression. INS , insulin gene.
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    Thermo Fisher dna free removal reagent
    INS sequence chromatographs for imprint analysis in the pouch young liver. Direct sequence analysis for INS in the pouch young liver. Chromatogram traces of genomic <t>DNA</t> (gDNA) from the mother and pouch young and of cDNA from the pouch young liver. ( A ) The pouch young inherited allele 2 (G-T) from its mother, and the clear monoallelic expression of allele 3 (A-T) in the liver was inherited from the father. ( B ) The pouch young inherited allele 1 (G-C) from its mother, and the clear monoallelic expression of allele 2 (G-T) in the liver was inherited from the father. <t>RNA</t> was extracted twice from the same liver sample and direct sequencing produced the same results in both samples. These results indicate that INS expression in the liver is a result of parent-of-origin specific genomic imprinting and not random monoallelic expression. INS , insulin gene.
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    Image Search Results


    Comparison of RNA snap ™ and Trizol® Max™ isolated RNA in an RT–PCR experiment. RNA isolated from SK4390 ( rph-1 ΔrppH ) was reversed transcribed with a primer specific for the lpp mRNA (∼330 nt) and subsequently PCR amplified for either 5,10,15, or 20 cycles (see ‘Materials and Methods’ section). The amplified PCR products were run on a 2% agarose gel and quantitated using ImageQuant TL software (GE). The amount of PCR product at the end of a fixed number of cycles from RNA snap ™ isolated RNA was set at 1 and compared with the amount of product obtained using Trizol® Max™ isolated RNA. Lanes 1 and 12, Gene Ruler™ Low Range DNA Ladder (Fermentas).

    Journal: Nucleic Acids Research

    Article Title: RNAsnap(TM): a rapid, quantitative and inexpensive, method for isolating total RNA from bacteria

    doi: 10.1093/nar/gks680

    Figure Lengend Snippet: Comparison of RNA snap ™ and Trizol® Max™ isolated RNA in an RT–PCR experiment. RNA isolated from SK4390 ( rph-1 ΔrppH ) was reversed transcribed with a primer specific for the lpp mRNA (∼330 nt) and subsequently PCR amplified for either 5,10,15, or 20 cycles (see ‘Materials and Methods’ section). The amplified PCR products were run on a 2% agarose gel and quantitated using ImageQuant TL software (GE). The amount of PCR product at the end of a fixed number of cycles from RNA snap ™ isolated RNA was set at 1 and compared with the amount of product obtained using Trizol® Max™ isolated RNA. Lanes 1 and 12, Gene Ruler™ Low Range DNA Ladder (Fermentas).

    Article Snippet: Both RNA samples were subjected to sodium acetate/ethanol precipitation, DNA removal with the DNA-free kit™ (Ambion) and a final sodium acetate/ethanol precipitation.

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Software

    Knockout of miR-UL148D impairs the establishment of experimental NR-1 latency in Kasumi-3 cells. (A, C) HCMV genome copies in Kasumi-3 cells (A) and CD34 + HPCs (C) infected with NR-1 or NR-1ΔmiR-UL148D. Total DNA was isolated from the infected cells at various time points after infection, and viral DNA was quantified by qPCR and normalized to cellular GAPDH. (B, D) Representative transcript levels from each class of viral genes in Kasumi-3 cells (B) and HPCs (D) infected with NR-1 or NR-1ΔmiR-UL148D. IE1 (immediately early), UL54 (early lytic transcript), and UL99 (late lytic transcript). Total RNA was isolated from infected cells and assayed by RT-qPCR. Samples were assayed in triplicate, and GAPDH level was used for normalization. (E, F) Restoring miR-UL148D expression via transfection with the miR-UL148D agomir reduced HCMV genome copies (E) and IE1 (F) expression in NR-1ΔmiR-UL148D-infected Kasumi-3 cells. The miR-UL148D agomir was added 24 hours before viral infection, and the culture media was replaced everyday with the addition of fresh agomir. DNA and total RNA were isolated from the Kasumi-3 cells at various time points after infection and quantified by qPCR and RT-qPCR, respectively. Samples were assayed in triplicate, and GAPDH level was used for normalization. Results derived from NR-1-infected Kasumi-3 cells are shown as a control. (G) Both NR-1ΔmiR-UL148D- and NR-1-infected Kasumi-3 cells produced infectious progeny. Infected Kasumi-3 cells harvested 10 days post-infection were stained with a monoclonal antibody against IE1 (clone 1B12, shown in red). GFP (green) and DAPI (blue) served as markers for lytic infection and nuclei, respectively. Infected Kasumi-3 cells were also co-cultured with HFFs. Viral plaque formation in the HFFs (shown by a GFP-positive status) was visualized by fluorescence microscopy. Images were collected using a 40x objective, and representative fields are shown for each infection. Values are shown as the mean ± SEM (n = 3). *, P

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus miR-UL148D Facilitates Latent Viral Infection by Targeting Host Cell Immediate Early Response Gene 5

    doi: 10.1371/journal.ppat.1006007

    Figure Lengend Snippet: Knockout of miR-UL148D impairs the establishment of experimental NR-1 latency in Kasumi-3 cells. (A, C) HCMV genome copies in Kasumi-3 cells (A) and CD34 + HPCs (C) infected with NR-1 or NR-1ΔmiR-UL148D. Total DNA was isolated from the infected cells at various time points after infection, and viral DNA was quantified by qPCR and normalized to cellular GAPDH. (B, D) Representative transcript levels from each class of viral genes in Kasumi-3 cells (B) and HPCs (D) infected with NR-1 or NR-1ΔmiR-UL148D. IE1 (immediately early), UL54 (early lytic transcript), and UL99 (late lytic transcript). Total RNA was isolated from infected cells and assayed by RT-qPCR. Samples were assayed in triplicate, and GAPDH level was used for normalization. (E, F) Restoring miR-UL148D expression via transfection with the miR-UL148D agomir reduced HCMV genome copies (E) and IE1 (F) expression in NR-1ΔmiR-UL148D-infected Kasumi-3 cells. The miR-UL148D agomir was added 24 hours before viral infection, and the culture media was replaced everyday with the addition of fresh agomir. DNA and total RNA were isolated from the Kasumi-3 cells at various time points after infection and quantified by qPCR and RT-qPCR, respectively. Samples were assayed in triplicate, and GAPDH level was used for normalization. Results derived from NR-1-infected Kasumi-3 cells are shown as a control. (G) Both NR-1ΔmiR-UL148D- and NR-1-infected Kasumi-3 cells produced infectious progeny. Infected Kasumi-3 cells harvested 10 days post-infection were stained with a monoclonal antibody against IE1 (clone 1B12, shown in red). GFP (green) and DAPI (blue) served as markers for lytic infection and nuclei, respectively. Infected Kasumi-3 cells were also co-cultured with HFFs. Viral plaque formation in the HFFs (shown by a GFP-positive status) was visualized by fluorescence microscopy. Images were collected using a 40x objective, and representative fields are shown for each infection. Values are shown as the mean ± SEM (n = 3). *, P

    Article Snippet: The RNA samples were treated with DNase using a DNA-free kit (Ambion, Austin, TX) according to the manufacturer’s instructions.

    Techniques: Knock-Out, Infection, Isolation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Transfection, Derivative Assay, Produced, Staining, Cell Culture, Fluorescence, Microscopy

    MiR-UL148D robustly accumulates in CD34 + progenitor cells during the establishment of experimental HCMV latency. (A) Kasumi-3 and CD34 + HPCs were efficiently infected with the NR-1 strain of HCMV. Kasumi-3 and HPCs were either mock-infected or infected with a GFP-expressing NR-1 strain at the indicated multiplicities of infection (MOIs). Two days later, the cells were analyzed for GFP expression by flow cytometry. An MOI of 5 was used for the following experiment. (B) The maintenance of the NR-1 genome, the suppression of viral IE1 and the presence of latency-associated UL138 over a 10-day time course. DNA and total RNA were isolated from Kasumi-3 cells and HPCs at various time points after infection. Viral genomic DNA was assayed by PCR, and RNA molecules encoding IE1 and UL138 were assayed by RT-PCR. In both cases, gel electrophoresis was used to detect the products of the reactions. (C) Reactivation of NR-1 virus in infected Kasumi-3 cells and HPCs. Kasumi-3 cells and HPCs were latently infected along a 10-day time course. Then, a subset of each cell population was cultured for an additional 2 days under conditions favoring lytic reactivation: Kasumi-3 cells were exposed to TPA, while HPCs were grown in reactivation medium. Following this, total RNA was extracted from the cells, and the ratio of IE1 to UL138 cDNA expression was assessed by qRT-PCR in triplicate. (D) Release of infectious progeny virions in latently infected Kasumi-3 cells and HPCs following reactivation treatment. Latently infected or mock-infected Kasumi-3 cells and HPCs were cultured under conditions favoring lytic reactivation (described above) or control conditions for 6 days, after which the cells were washed with PBS and co-cultured with HFFs for 2 days. Then, the Kasumi-3 cells were removed from the co-cultures, and the HFFs were washed with PBS and cultured for an additional 5 days for fluorescence microscopy analysis of GFP-positive plaques. (E) miR-UL148D showed robust accumulation during the establishment of experimental HCMV latency in Kasumi-3 cells. In total, 20,000 infected cells were harvested for the isolation of total RNA and DNA at each indicted time point along the 10-day time course. Viral DNA was first quantified by qPCR, and then absolute viral genomes copies were calculated by generating a standard curve. HCMV miRNAs were then assayed with a HCMV miRNA probe kit, and their levels were calculated using a standard curve. The HCMV miRNA level per virus was calculated by dividing the amount of each HCMV miRNA by the virus copy number. (F) miR-UL148D accumulated in HPCs latently infected with NR-1. HCMV miRNA levels in NR-1-infected HPCs were determined as described above. Values are shown as the mean ± SEM (n = 3). **, P

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus miR-UL148D Facilitates Latent Viral Infection by Targeting Host Cell Immediate Early Response Gene 5

    doi: 10.1371/journal.ppat.1006007

    Figure Lengend Snippet: MiR-UL148D robustly accumulates in CD34 + progenitor cells during the establishment of experimental HCMV latency. (A) Kasumi-3 and CD34 + HPCs were efficiently infected with the NR-1 strain of HCMV. Kasumi-3 and HPCs were either mock-infected or infected with a GFP-expressing NR-1 strain at the indicated multiplicities of infection (MOIs). Two days later, the cells were analyzed for GFP expression by flow cytometry. An MOI of 5 was used for the following experiment. (B) The maintenance of the NR-1 genome, the suppression of viral IE1 and the presence of latency-associated UL138 over a 10-day time course. DNA and total RNA were isolated from Kasumi-3 cells and HPCs at various time points after infection. Viral genomic DNA was assayed by PCR, and RNA molecules encoding IE1 and UL138 were assayed by RT-PCR. In both cases, gel electrophoresis was used to detect the products of the reactions. (C) Reactivation of NR-1 virus in infected Kasumi-3 cells and HPCs. Kasumi-3 cells and HPCs were latently infected along a 10-day time course. Then, a subset of each cell population was cultured for an additional 2 days under conditions favoring lytic reactivation: Kasumi-3 cells were exposed to TPA, while HPCs were grown in reactivation medium. Following this, total RNA was extracted from the cells, and the ratio of IE1 to UL138 cDNA expression was assessed by qRT-PCR in triplicate. (D) Release of infectious progeny virions in latently infected Kasumi-3 cells and HPCs following reactivation treatment. Latently infected or mock-infected Kasumi-3 cells and HPCs were cultured under conditions favoring lytic reactivation (described above) or control conditions for 6 days, after which the cells were washed with PBS and co-cultured with HFFs for 2 days. Then, the Kasumi-3 cells were removed from the co-cultures, and the HFFs were washed with PBS and cultured for an additional 5 days for fluorescence microscopy analysis of GFP-positive plaques. (E) miR-UL148D showed robust accumulation during the establishment of experimental HCMV latency in Kasumi-3 cells. In total, 20,000 infected cells were harvested for the isolation of total RNA and DNA at each indicted time point along the 10-day time course. Viral DNA was first quantified by qPCR, and then absolute viral genomes copies were calculated by generating a standard curve. HCMV miRNAs were then assayed with a HCMV miRNA probe kit, and their levels were calculated using a standard curve. The HCMV miRNA level per virus was calculated by dividing the amount of each HCMV miRNA by the virus copy number. (F) miR-UL148D accumulated in HPCs latently infected with NR-1. HCMV miRNA levels in NR-1-infected HPCs were determined as described above. Values are shown as the mean ± SEM (n = 3). **, P

    Article Snippet: The RNA samples were treated with DNase using a DNA-free kit (Ambion, Austin, TX) according to the manufacturer’s instructions.

    Techniques: Infection, Expressing, Flow Cytometry, Cytometry, Isolation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Cell Culture, Quantitative RT-PCR, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction

    CDC25 expression is essential for silencing IE1 transcription. (A, B) Stable overexpression of IER5 induced viral lytic gene transcription in NR-1-infected Kasumi-3 cells. IER5 was stably overexpressed in Kasumi-3 cells infected with LV-IER5. An empty-backbone lentivirus was used as a control. The cells were then infected with NR-1 virus for a 10-day time course. The infected cells were analyzed for IER5 and CDC25B protein expression (A) and IE1 (immediately early), UL54 (early lytic transcript), and UL99 (late lytic transcript) mRNA expression (B). (C, D) Stable overexpression of CDC25B suppressed viral lytic gene transcription in NR-1ΔmiR-UL148D-infected Kasumi-3 cells. CDC25B was also stably overexpressed in Kasumi-3 cells infected with LV-CDC25B. An empty-backbone lentivirus was used as a control. The cells were then infected with NR-1 or NR-1ΔmiR-UL148D virus for a 10-day time course. IER5 and CDC25B protein expression (C) and IE1, UL54, and UL99 mRNA expression (D) were then measured in the Kasumi-3 cells. (E) Pharmaceutical inhibition of CDC25B efficiently induced viral lytic gene transcription in NR-1-infected cells. After infection with NR-1, Kasumi-3 cells were incubated with NSC663284, a specific CDC25B inhibitor, at a concentration of 5 μM for 48 hours. Then, the treated cells were further cultured in fresh media for 5 days. The DNA content in the treated cells was analyzed 2 dpi. Total RNA was extracted for RT-qPCR analysis of IE1, UL54 and UL99 expression at the indicated time points along a 7-day time course. Values are shown as the mean ± SEM (n = 3). **, P

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus miR-UL148D Facilitates Latent Viral Infection by Targeting Host Cell Immediate Early Response Gene 5

    doi: 10.1371/journal.ppat.1006007

    Figure Lengend Snippet: CDC25 expression is essential for silencing IE1 transcription. (A, B) Stable overexpression of IER5 induced viral lytic gene transcription in NR-1-infected Kasumi-3 cells. IER5 was stably overexpressed in Kasumi-3 cells infected with LV-IER5. An empty-backbone lentivirus was used as a control. The cells were then infected with NR-1 virus for a 10-day time course. The infected cells were analyzed for IER5 and CDC25B protein expression (A) and IE1 (immediately early), UL54 (early lytic transcript), and UL99 (late lytic transcript) mRNA expression (B). (C, D) Stable overexpression of CDC25B suppressed viral lytic gene transcription in NR-1ΔmiR-UL148D-infected Kasumi-3 cells. CDC25B was also stably overexpressed in Kasumi-3 cells infected with LV-CDC25B. An empty-backbone lentivirus was used as a control. The cells were then infected with NR-1 or NR-1ΔmiR-UL148D virus for a 10-day time course. IER5 and CDC25B protein expression (C) and IE1, UL54, and UL99 mRNA expression (D) were then measured in the Kasumi-3 cells. (E) Pharmaceutical inhibition of CDC25B efficiently induced viral lytic gene transcription in NR-1-infected cells. After infection with NR-1, Kasumi-3 cells were incubated with NSC663284, a specific CDC25B inhibitor, at a concentration of 5 μM for 48 hours. Then, the treated cells were further cultured in fresh media for 5 days. The DNA content in the treated cells was analyzed 2 dpi. Total RNA was extracted for RT-qPCR analysis of IE1, UL54 and UL99 expression at the indicated time points along a 7-day time course. Values are shown as the mean ± SEM (n = 3). **, P

    Article Snippet: The RNA samples were treated with DNase using a DNA-free kit (Ambion, Austin, TX) according to the manufacturer’s instructions.

    Techniques: Expressing, Over Expression, Infection, Stable Transfection, Inhibition, Incubation, Concentration Assay, Cell Culture, Quantitative RT-PCR

    CX-5461 treatment disrupts HCMV replication at the stage of viral DNA synthesis (A) MRC-5 fibroblasts were infected with TB40/E virus or mock infected at an MOI of 3 IU/cell. Cells were treated with 0.5 μM CX-5461 or vehicle control at 2 or 48 hpi as indicated. Total RNA was isolated at the indicated times and IE1 and GAPDH levels were determined by quantitative RT-PCR. (B) Under the same conditions, viral DNA levels were determined using quantitative PCR and primers against UL123 and GAPDH. (C) Fibroblasts were infected as above and treated starting at 48 hpi. Whole cell lysates were collected at multiple times post infection and analyzed by Western blot using antibodies against the indicated proteins. (D) Samples were fixed 96 hpi and stained using the indicated antibody. Arrows identify two examples of disrupted replication compartments. Quantitative PCR data are from three biological replicates and two technical replicates with error bars representing standard deviation from the mean.

    Journal: Antiviral research

    Article Title: Impact of RNA polymerase I inhibitor CX-5461 on viral kinase-dependent and -independent cytomegalovirus replication

    doi: 10.1016/j.antiviral.2018.02.014

    Figure Lengend Snippet: CX-5461 treatment disrupts HCMV replication at the stage of viral DNA synthesis (A) MRC-5 fibroblasts were infected with TB40/E virus or mock infected at an MOI of 3 IU/cell. Cells were treated with 0.5 μM CX-5461 or vehicle control at 2 or 48 hpi as indicated. Total RNA was isolated at the indicated times and IE1 and GAPDH levels were determined by quantitative RT-PCR. (B) Under the same conditions, viral DNA levels were determined using quantitative PCR and primers against UL123 and GAPDH. (C) Fibroblasts were infected as above and treated starting at 48 hpi. Whole cell lysates were collected at multiple times post infection and analyzed by Western blot using antibodies against the indicated proteins. (D) Samples were fixed 96 hpi and stained using the indicated antibody. Arrows identify two examples of disrupted replication compartments. Quantitative PCR data are from three biological replicates and two technical replicates with error bars representing standard deviation from the mean.

    Article Snippet: Approximately 2 μg of RNA was treated with DNA-free DNA Removal Kit (ThermoFisher Scientific) and used to synthesize cDNA with random hexamers and Superscript III Reverse Transcriptase (ThermoFisher Scientific). qPCR was performed as described using primers against CMV UL123, GAPDH and pre-rRNA external transcribed spacer 1 (ETS) (5′-GAACGGTGGTGTGTCGTTC-3′ and 5′-GCGTCTCGTCTCGTCTCACT-3′).

    Techniques: DNA Synthesis, Infection, Isolation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot, Staining, Standard Deviation

    Expression of transcripts for toll-like receptors (TLR) in TIGKs RT-PCR of TIGK mRNA from two separate experiments, superscript 1 and 2, using primers for the TLRs indicated. Genomic DNA templates serve as positive controls (PC), and negative controls are DNase I-treated total RNA (R). Each template cDNA was run in duplicate reactions for each experiment.

    Journal: Journal of periodontal research

    Article Title: Establishment and characterization of a telomerase immortalized human gingival epithelial cell line

    doi: 10.1111/jre.12059

    Figure Lengend Snippet: Expression of transcripts for toll-like receptors (TLR) in TIGKs RT-PCR of TIGK mRNA from two separate experiments, superscript 1 and 2, using primers for the TLRs indicated. Genomic DNA templates serve as positive controls (PC), and negative controls are DNase I-treated total RNA (R). Each template cDNA was run in duplicate reactions for each experiment.

    Article Snippet: Briefly, cell cultures were homogenized directly in TRIzol® Reagent (Invitrogen), total RNA isolated using standard protocols and then treated with DNase I using Ambion’s DNA- free ™ Reagent Kit (Applied Biosystems, Foster City, CA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    INS sequence chromatographs for imprint analysis in the pouch young liver. Direct sequence analysis for INS in the pouch young liver. Chromatogram traces of genomic DNA (gDNA) from the mother and pouch young and of cDNA from the pouch young liver. ( A ) The pouch young inherited allele 2 (G-T) from its mother, and the clear monoallelic expression of allele 3 (A-T) in the liver was inherited from the father. ( B ) The pouch young inherited allele 1 (G-C) from its mother, and the clear monoallelic expression of allele 2 (G-T) in the liver was inherited from the father. RNA was extracted twice from the same liver sample and direct sequencing produced the same results in both samples. These results indicate that INS expression in the liver is a result of parent-of-origin specific genomic imprinting and not random monoallelic expression. INS , insulin gene.

    Journal: Epigenetics & Chromatin

    Article Title: Selected imprinting of INS in the marsupial

    doi: 10.1186/1756-8935-5-14

    Figure Lengend Snippet: INS sequence chromatographs for imprint analysis in the pouch young liver. Direct sequence analysis for INS in the pouch young liver. Chromatogram traces of genomic DNA (gDNA) from the mother and pouch young and of cDNA from the pouch young liver. ( A ) The pouch young inherited allele 2 (G-T) from its mother, and the clear monoallelic expression of allele 3 (A-T) in the liver was inherited from the father. ( B ) The pouch young inherited allele 1 (G-C) from its mother, and the clear monoallelic expression of allele 2 (G-T) in the liver was inherited from the father. RNA was extracted twice from the same liver sample and direct sequencing produced the same results in both samples. These results indicate that INS expression in the liver is a result of parent-of-origin specific genomic imprinting and not random monoallelic expression. INS , insulin gene.

    Article Snippet: Total RNA was DNase treated (DNA-free™; Ambion) to remove contaminating DNA, run on a 1% agarose gel to assess the quality, quantified with a nano-spectrometer (NanoDrop ND-1000 Spectrophotometer; NanoDrop Technologies Inc., Wilmington, DE, USA) and cDNA was synthesised using the SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen, Carisbad, CA, USA).

    Techniques: Sequencing, Expressing, Produced

    Structure and methylation of tammar INS. ( A ) 5 ′ -Rapid amplification of cDNA ends (5 ′ -RACE) was performed on RNA derived from one pancreas (Panc), two mammary glands (MG) and one liver (Liv). Five INS transcripts were amplified using a primer designed in the first INS coding exon (half-arrow). Three transcripts were chimeras and contained an exon derived from the neighbouring tyrosine hydroxylase ( TH ) gene and two were transcribed from the INS noncoding exon. The mammary gland 1 (MG1; lactation phase 1) and liver expressed both types of transcripts, the pancreas expressed only the INS-derived transcripts, and the mammary gland 2 (MG2; lactation phase 3) expressed only the TH-INS transcripts. ( B ) Schematic of predicted tammar TH and INS genes (not to scale). Predicted coding exons (grey), verified coding exons (black) and noncoding exons (white) are represented by boxes. Transcription start sites identified by 5 ′ -RACE are indicated with turned arrows. CpGs are indicated by short vertical black lines. SNPs are indicated by black triangles. Bisulphite sequenced regions (black horizontal lines) are shown with individual bisulphite sequences underneath: open and closed circles are unmethylated and methylated CpGs, respectively. Each row represents the methylation pattern on a separate DNA fragment from the same sample. Both methylated and unmethylated alleles were present in the liver and mammary gland tissues at the TH-INS TSS. Only methylated alleles were present at the CpG Island and the INS TSS had a variable methylation pattern. INS , insulin gene.

    Journal: Epigenetics & Chromatin

    Article Title: Selected imprinting of INS in the marsupial

    doi: 10.1186/1756-8935-5-14

    Figure Lengend Snippet: Structure and methylation of tammar INS. ( A ) 5 ′ -Rapid amplification of cDNA ends (5 ′ -RACE) was performed on RNA derived from one pancreas (Panc), two mammary glands (MG) and one liver (Liv). Five INS transcripts were amplified using a primer designed in the first INS coding exon (half-arrow). Three transcripts were chimeras and contained an exon derived from the neighbouring tyrosine hydroxylase ( TH ) gene and two were transcribed from the INS noncoding exon. The mammary gland 1 (MG1; lactation phase 1) and liver expressed both types of transcripts, the pancreas expressed only the INS-derived transcripts, and the mammary gland 2 (MG2; lactation phase 3) expressed only the TH-INS transcripts. ( B ) Schematic of predicted tammar TH and INS genes (not to scale). Predicted coding exons (grey), verified coding exons (black) and noncoding exons (white) are represented by boxes. Transcription start sites identified by 5 ′ -RACE are indicated with turned arrows. CpGs are indicated by short vertical black lines. SNPs are indicated by black triangles. Bisulphite sequenced regions (black horizontal lines) are shown with individual bisulphite sequences underneath: open and closed circles are unmethylated and methylated CpGs, respectively. Each row represents the methylation pattern on a separate DNA fragment from the same sample. Both methylated and unmethylated alleles were present in the liver and mammary gland tissues at the TH-INS TSS. Only methylated alleles were present at the CpG Island and the INS TSS had a variable methylation pattern. INS , insulin gene.

    Article Snippet: Total RNA was DNase treated (DNA-free™; Ambion) to remove contaminating DNA, run on a 1% agarose gel to assess the quality, quantified with a nano-spectrometer (NanoDrop ND-1000 Spectrophotometer; NanoDrop Technologies Inc., Wilmington, DE, USA) and cDNA was synthesised using the SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen, Carisbad, CA, USA).

    Techniques: Methylation, Rapid Amplification of cDNA Ends, Derivative Assay, Amplification