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    Qiagen rneasy mini kit
    Stimulation of an IFN-mediated antiviral response in human transformed or tumor cells upon Poly(I:C) transfection. ( A ) HEK293, HEK293T, NB324K and Hela cultures were <t>transfected</t> with 2 µg/ml of synthetic dsRNA Poly(I:C) (pI:C) or a 150 mM NaCl solution as control, using lipofectamine 2000 for the period indicated in the figure. The respective culture media were then collected, centrifuged to discard cellular debris, and analyzed by Enzyme-linked Immuno-Sorbent Assay (ELISA) for their content in human IFN-β. Each result is represented as mean+standard deviation of three independent experiments. ( B ) Expression of IFN-α and IFN-β transcripts in mock-treated or pI:C-transfected human cell lines was assessed by RT-PCRs. At the indicated time points, total RNAs were extracted from the respective cultures using the <t>RNeasy</t> kit. One µg of RNA was then treated by DNase I in order to remove potential contaminating DNA and reverse transcribed into cDNA. A fraction of the obtained cDNA (1/10) was then analyzed by PCR for its content in type-I IFN transcripts using specific primer pairs. Transcripts encoding the Human 18S ribosomal protein were used as internal loading controls. Data shown are representative of 3 experiments which gave similar results. ( C ) Assessment of the IFN-signaling (Jak/STAT) pathway activation in HEK293, HEK293T, NB324K and Hela cells upon pI:C transfection. At the time indicated mock-treated or pI:C-transfected (2 µg/ml) cultures were harvested by scraping in PBS and centrifuged. Cell pellets were then re-suspended in complete Ripa buffer supplemented with phosphatase and protease inhibitors. Total proteins were extracted from each sample as described in Materials and Methods . Fifty µg total proteins per sample were then subjected to bipartite 8/10% SDS-PAGE, transferred onto membranes, and probed with antibodies specific for phosphorylated and total isoforms of STAT 1 and STAT 2 as well as with an antibody specific to PKR. Actin was used as an internal loading control. Each presented blot is representative of 3 additional which gave similar results.
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    Stimulation of an IFN-mediated antiviral response in human transformed or tumor cells upon Poly(I:C) transfection. ( A ) HEK293, HEK293T, NB324K and Hela cultures were transfected with 2 µg/ml of synthetic dsRNA Poly(I:C) (pI:C) or a 150 mM NaCl solution as control, using lipofectamine 2000 for the period indicated in the figure. The respective culture media were then collected, centrifuged to discard cellular debris, and analyzed by Enzyme-linked Immuno-Sorbent Assay (ELISA) for their content in human IFN-β. Each result is represented as mean+standard deviation of three independent experiments. ( B ) Expression of IFN-α and IFN-β transcripts in mock-treated or pI:C-transfected human cell lines was assessed by RT-PCRs. At the indicated time points, total RNAs were extracted from the respective cultures using the RNeasy kit. One µg of RNA was then treated by DNase I in order to remove potential contaminating DNA and reverse transcribed into cDNA. A fraction of the obtained cDNA (1/10) was then analyzed by PCR for its content in type-I IFN transcripts using specific primer pairs. Transcripts encoding the Human 18S ribosomal protein were used as internal loading controls. Data shown are representative of 3 experiments which gave similar results. ( C ) Assessment of the IFN-signaling (Jak/STAT) pathway activation in HEK293, HEK293T, NB324K and Hela cells upon pI:C transfection. At the time indicated mock-treated or pI:C-transfected (2 µg/ml) cultures were harvested by scraping in PBS and centrifuged. Cell pellets were then re-suspended in complete Ripa buffer supplemented with phosphatase and protease inhibitors. Total proteins were extracted from each sample as described in Materials and Methods . Fifty µg total proteins per sample were then subjected to bipartite 8/10% SDS-PAGE, transferred onto membranes, and probed with antibodies specific for phosphorylated and total isoforms of STAT 1 and STAT 2 as well as with an antibody specific to PKR. Actin was used as an internal loading control. Each presented blot is representative of 3 additional which gave similar results.

    Journal: PLoS ONE

    Article Title: TLR-9 Contributes to the Antiviral Innate Immune Sensing of Rodent Parvoviruses MVMp and H-1PV by Normal Human Immune Cells

    doi: 10.1371/journal.pone.0055086

    Figure Lengend Snippet: Stimulation of an IFN-mediated antiviral response in human transformed or tumor cells upon Poly(I:C) transfection. ( A ) HEK293, HEK293T, NB324K and Hela cultures were transfected with 2 µg/ml of synthetic dsRNA Poly(I:C) (pI:C) or a 150 mM NaCl solution as control, using lipofectamine 2000 for the period indicated in the figure. The respective culture media were then collected, centrifuged to discard cellular debris, and analyzed by Enzyme-linked Immuno-Sorbent Assay (ELISA) for their content in human IFN-β. Each result is represented as mean+standard deviation of three independent experiments. ( B ) Expression of IFN-α and IFN-β transcripts in mock-treated or pI:C-transfected human cell lines was assessed by RT-PCRs. At the indicated time points, total RNAs were extracted from the respective cultures using the RNeasy kit. One µg of RNA was then treated by DNase I in order to remove potential contaminating DNA and reverse transcribed into cDNA. A fraction of the obtained cDNA (1/10) was then analyzed by PCR for its content in type-I IFN transcripts using specific primer pairs. Transcripts encoding the Human 18S ribosomal protein were used as internal loading controls. Data shown are representative of 3 experiments which gave similar results. ( C ) Assessment of the IFN-signaling (Jak/STAT) pathway activation in HEK293, HEK293T, NB324K and Hela cells upon pI:C transfection. At the time indicated mock-treated or pI:C-transfected (2 µg/ml) cultures were harvested by scraping in PBS and centrifuged. Cell pellets were then re-suspended in complete Ripa buffer supplemented with phosphatase and protease inhibitors. Total proteins were extracted from each sample as described in Materials and Methods . Fifty µg total proteins per sample were then subjected to bipartite 8/10% SDS-PAGE, transferred onto membranes, and probed with antibodies specific for phosphorylated and total isoforms of STAT 1 and STAT 2 as well as with an antibody specific to PKR. Actin was used as an internal loading control. Each presented blot is representative of 3 additional which gave similar results.

    Article Snippet: Briefly, Total RNAs of mock-treated, parvovirus- or NDV-infected, and/or PolyI:C transfected cells were isolated using an RNeasy mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.

    Techniques: Transformation Assay, Transfection, Enzyme-linked Immunosorbent Assay, Standard Deviation, Expressing, Polymerase Chain Reaction, Activation Assay, SDS Page

    Time-dependent transcriptional up-regulation of type-I IFN encoding mRNAs in MVMp- or H-1PV-infected human cell lines. ( A ) HEK293 and HEK293T, ( B ) NB324K and ( C ) Hela cells were mock-treated for 24 hrs, infected with parvoviruses (5 PFUs/cell) for the indicated times, or infected with NDV (6 HU/10 6 cells) or transfected with pI:C (2 µg/ml) for 15 hrs. At the indicated times, cells were harvested and total RNAs were extracted using the RNeasy kit. One µg was then reverse transcribed into cDNA and 10% of this product was subjected to PCR reactions using sets of primers specific to each indicated cytokine mRNA or viral transcripts. PCR product of 18S ribosomal RNA was used as housekeeping gene to normalize loading. No signal was detected in the samples when omitting the reverse transcriptase. Presented data are representative of 3 experiments which all gave similar results.

    Journal: PLoS ONE

    Article Title: TLR-9 Contributes to the Antiviral Innate Immune Sensing of Rodent Parvoviruses MVMp and H-1PV by Normal Human Immune Cells

    doi: 10.1371/journal.pone.0055086

    Figure Lengend Snippet: Time-dependent transcriptional up-regulation of type-I IFN encoding mRNAs in MVMp- or H-1PV-infected human cell lines. ( A ) HEK293 and HEK293T, ( B ) NB324K and ( C ) Hela cells were mock-treated for 24 hrs, infected with parvoviruses (5 PFUs/cell) for the indicated times, or infected with NDV (6 HU/10 6 cells) or transfected with pI:C (2 µg/ml) for 15 hrs. At the indicated times, cells were harvested and total RNAs were extracted using the RNeasy kit. One µg was then reverse transcribed into cDNA and 10% of this product was subjected to PCR reactions using sets of primers specific to each indicated cytokine mRNA or viral transcripts. PCR product of 18S ribosomal RNA was used as housekeeping gene to normalize loading. No signal was detected in the samples when omitting the reverse transcriptase. Presented data are representative of 3 experiments which all gave similar results.

    Article Snippet: Briefly, Total RNAs of mock-treated, parvovirus- or NDV-infected, and/or PolyI:C transfected cells were isolated using an RNeasy mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.

    Techniques: Infection, Transfection, Polymerase Chain Reaction

    Activation of both IFN-producing and IFN-signaling pathways in hPBMCs upon parvovirus infection and comparison of their intensity to that triggered by NDV. ( A , B and D ) hPBMCs collected from the blood of healthy donors were distributed into 6-well plates at 1×10 7 cells/5 ml culture medium/well. They were then mock-treated or infected with MVMp or H-1PV at 20 PFUs/cell or with NDV (6 HU/10 6 cells). Cultures were harvested in PBS and centrifuged at the time p.i. indicated in each figure. ( A , D ) One half of each pellet was resuspended in Ripa buffer supplemented with phosphatase and protease inhibitors in order to perform Western blot experiments whereas ( B ) total RNAs were extracted from the rest of each cell pellet using the RNeasy kit. ( A , D ) Total proteins were extracted from each sample as described in Materials and Methods . Seventy µg total proteins per sample were then subjected to 10% SDS-PAGE, transferred onto membranes, and probed with antibodies specific for total and phosphorylated STAT 2 and STAT 1 polypeptides as well as for PKR. Actin was used as an internal loading control. Each presented blot is representative of 4 additional which gave similar results. ( B ). One µg of isolated total RNA was then reverse transcribed into cDNA and 10% of this product was subjected to PCR reactions using sets of primers specific to each indicated mRNA. PCR product of 18S ribosomal RNA was used as housekeeping gene to normalize loading. No signal was detected in the samples when omitting the reverse transcriptase. Presented data are representative of 4 experiments which all gave similar results. ( C ) hPBMCs collected from the blood of healthy donors were distributed into 24-well plates at 1×10 6 cells/500 µl culture medium/well. They were then mock-treated or infected with MVMp, H-1PV (20 PFUs/cell) or NDV (6 HU/10 6 cells). After a period of incubation of 24 hrs media were collected, centrifuged in order to discard cellular debris and analyzed by Enzyme-linked Immuno-Sorbent Assay (ELISA) for their content in IFN-α and IFN-β. Results are expressed as means+standard deviations of seven independent experiments.

    Journal: PLoS ONE

    Article Title: TLR-9 Contributes to the Antiviral Innate Immune Sensing of Rodent Parvoviruses MVMp and H-1PV by Normal Human Immune Cells

    doi: 10.1371/journal.pone.0055086

    Figure Lengend Snippet: Activation of both IFN-producing and IFN-signaling pathways in hPBMCs upon parvovirus infection and comparison of their intensity to that triggered by NDV. ( A , B and D ) hPBMCs collected from the blood of healthy donors were distributed into 6-well plates at 1×10 7 cells/5 ml culture medium/well. They were then mock-treated or infected with MVMp or H-1PV at 20 PFUs/cell or with NDV (6 HU/10 6 cells). Cultures were harvested in PBS and centrifuged at the time p.i. indicated in each figure. ( A , D ) One half of each pellet was resuspended in Ripa buffer supplemented with phosphatase and protease inhibitors in order to perform Western blot experiments whereas ( B ) total RNAs were extracted from the rest of each cell pellet using the RNeasy kit. ( A , D ) Total proteins were extracted from each sample as described in Materials and Methods . Seventy µg total proteins per sample were then subjected to 10% SDS-PAGE, transferred onto membranes, and probed with antibodies specific for total and phosphorylated STAT 2 and STAT 1 polypeptides as well as for PKR. Actin was used as an internal loading control. Each presented blot is representative of 4 additional which gave similar results. ( B ). One µg of isolated total RNA was then reverse transcribed into cDNA and 10% of this product was subjected to PCR reactions using sets of primers specific to each indicated mRNA. PCR product of 18S ribosomal RNA was used as housekeeping gene to normalize loading. No signal was detected in the samples when omitting the reverse transcriptase. Presented data are representative of 4 experiments which all gave similar results. ( C ) hPBMCs collected from the blood of healthy donors were distributed into 24-well plates at 1×10 6 cells/500 µl culture medium/well. They were then mock-treated or infected with MVMp, H-1PV (20 PFUs/cell) or NDV (6 HU/10 6 cells). After a period of incubation of 24 hrs media were collected, centrifuged in order to discard cellular debris and analyzed by Enzyme-linked Immuno-Sorbent Assay (ELISA) for their content in IFN-α and IFN-β. Results are expressed as means+standard deviations of seven independent experiments.

    Article Snippet: Briefly, Total RNAs of mock-treated, parvovirus- or NDV-infected, and/or PolyI:C transfected cells were isolated using an RNeasy mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.

    Techniques: Activation Assay, Infection, Western Blot, SDS Page, Isolation, Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay

    Activation of a TLR-9 dependent production of type-I IFNs in parvovirus-infected human Namalwa cells. The cells (1.10 6 cells/well of a 24-well plate) were either infected with MVMp or H-1PV (∼80 PFUs/cell equivalent to 1×10 4 virus genomes/cell), stimulated with the TLR-9 agonist ODN 2395 at 1 µM, or mock-treated. ( A ) At the indicated time points culture supernatants were collected from the respective wells and used to determine the quantity of type-I IFNs released in the culture medium by ELISA experiments. Results are expressed as means+standard deviations of three independent experiments. ( B ) Mock-treated, parvovirus-infected or ODN stimulated Namalwa cells were harvested at the indicated time points and total RNAs were extracted using the RNeasy kit as described previously in Figure 1 . Total RNAs extracted from parvovirus-infected (24 hrs p.i. at 80 PFUs/cell) or pI:C-transfected (15 hrs post-transfection with 2 µg dsRNA/ml) NB324K cells were used as positive or negative controls. Presented data are representative of 3 experiments which all gave similar results. ( C ) Total DNA was harvested at the indicated time points from parvovirus-infected (MOI of 80 PFUs/cell) or mock-treated Namalwa or HEK293T cultures to assess by Southern blot experiments as described in Figure 2 the replication of both parvoviruses (dRF, dimmer replicative form; mRF, monomeric replicative form; ssDNA, single-stranded DNA genome). The blot shown is representative of 3 experiments which gave similar results. ( D ) Cell pellets from mock-treated or parvovirus-infected Namalwa cultures were re-suspended in complete Ripa buffer and Western blotting was performed as described in Figure 6 . Actin was used as an internal loading control. Each presented blot is representative of 3 experiments which gave similar results.

    Journal: PLoS ONE

    Article Title: TLR-9 Contributes to the Antiviral Innate Immune Sensing of Rodent Parvoviruses MVMp and H-1PV by Normal Human Immune Cells

    doi: 10.1371/journal.pone.0055086

    Figure Lengend Snippet: Activation of a TLR-9 dependent production of type-I IFNs in parvovirus-infected human Namalwa cells. The cells (1.10 6 cells/well of a 24-well plate) were either infected with MVMp or H-1PV (∼80 PFUs/cell equivalent to 1×10 4 virus genomes/cell), stimulated with the TLR-9 agonist ODN 2395 at 1 µM, or mock-treated. ( A ) At the indicated time points culture supernatants were collected from the respective wells and used to determine the quantity of type-I IFNs released in the culture medium by ELISA experiments. Results are expressed as means+standard deviations of three independent experiments. ( B ) Mock-treated, parvovirus-infected or ODN stimulated Namalwa cells were harvested at the indicated time points and total RNAs were extracted using the RNeasy kit as described previously in Figure 1 . Total RNAs extracted from parvovirus-infected (24 hrs p.i. at 80 PFUs/cell) or pI:C-transfected (15 hrs post-transfection with 2 µg dsRNA/ml) NB324K cells were used as positive or negative controls. Presented data are representative of 3 experiments which all gave similar results. ( C ) Total DNA was harvested at the indicated time points from parvovirus-infected (MOI of 80 PFUs/cell) or mock-treated Namalwa or HEK293T cultures to assess by Southern blot experiments as described in Figure 2 the replication of both parvoviruses (dRF, dimmer replicative form; mRF, monomeric replicative form; ssDNA, single-stranded DNA genome). The blot shown is representative of 3 experiments which gave similar results. ( D ) Cell pellets from mock-treated or parvovirus-infected Namalwa cultures were re-suspended in complete Ripa buffer and Western blotting was performed as described in Figure 6 . Actin was used as an internal loading control. Each presented blot is representative of 3 experiments which gave similar results.

    Article Snippet: Briefly, Total RNAs of mock-treated, parvovirus- or NDV-infected, and/or PolyI:C transfected cells were isolated using an RNeasy mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.

    Techniques: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Transfection, Southern Blot, Western Blot

    Comparison of sera exosomal RNA using four different RNA extraction methods. (A) Total RNA yield from ultracentrifugation (UC) and ExoQuick (EQ) treated samples using the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and AllPrep DNA/RNA Mini Kit. (B) Demonstration of RNA quality measured by OD 260 /OD 280 in EQ and UC treated samples. Data are shown as the mean ± SD from six independent patient samples. *** P

    Journal: PLoS ONE

    Article Title: Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens

    doi: 10.1371/journal.pone.0196913

    Figure Lengend Snippet: Comparison of sera exosomal RNA using four different RNA extraction methods. (A) Total RNA yield from ultracentrifugation (UC) and ExoQuick (EQ) treated samples using the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and AllPrep DNA/RNA Mini Kit. (B) Demonstration of RNA quality measured by OD 260 /OD 280 in EQ and UC treated samples. Data are shown as the mean ± SD from six independent patient samples. *** P

    Article Snippet: RNA yield and OD260 /OD280 ratios were therefore compared for exosomal RNA isolated using four different RNA extraction methods: the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and the AllPrep DNA/RNA Mini Kit ( ).

    Techniques: RNA Extraction

    Exosomal RNAs are stable over two decades. Dot plots of total RNA yield and OD 260 /OD 280 from 105 EQ-treated archival patient samples using the RNeasy Mini Kit combined with TRIzol LS. Storage time for each sample is indicated on the x-axis. The Spearman’s rank correlations for RNA yield versus storing time and OD 260 /OD 280 versus storage time are 0.185 ( P = 0.06) and 0.04 ( P = 0.70), respectively.

    Journal: PLoS ONE

    Article Title: Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens

    doi: 10.1371/journal.pone.0196913

    Figure Lengend Snippet: Exosomal RNAs are stable over two decades. Dot plots of total RNA yield and OD 260 /OD 280 from 105 EQ-treated archival patient samples using the RNeasy Mini Kit combined with TRIzol LS. Storage time for each sample is indicated on the x-axis. The Spearman’s rank correlations for RNA yield versus storing time and OD 260 /OD 280 versus storage time are 0.185 ( P = 0.06) and 0.04 ( P = 0.70), respectively.

    Article Snippet: RNA yield and OD260 /OD280 ratios were therefore compared for exosomal RNA isolated using four different RNA extraction methods: the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and the AllPrep DNA/RNA Mini Kit ( ).

    Techniques:

    Quantitative gene expression of PLLA/HA at different time points during differentiation process. (a) Gene expression of beta-catenin, frizzled, alkaline phosphatase, and osteopontin. (b) OC (BGALP), osteonectin, Runx2, and Col. (c) Bone sialoprotein, integrin, bone morphogenic protein 2, PP2A (serine/threonine phosphatase), and c-fos. The total RNA was extracted from hMSCs cultured on the substrates (n = 6) at different time points (0, 7, 14, and 21 days) using the RNeasy Mini kit (Qiagen, Chatsworth, CA, USA). Following cDNA synthesis and qPCR, the relative gene expression was normalized to GAPDH and baseline expression. The P value was set at level

    Journal: PLoS ONE

    Article Title: A Comparative Study on In Vitro Osteogenic Priming Potential of Electron Spun Scaffold PLLA/HA/Col, PLLA/HA, and PLLA/Col for Tissue Engineering Application

    doi: 10.1371/journal.pone.0104389

    Figure Lengend Snippet: Quantitative gene expression of PLLA/HA at different time points during differentiation process. (a) Gene expression of beta-catenin, frizzled, alkaline phosphatase, and osteopontin. (b) OC (BGALP), osteonectin, Runx2, and Col. (c) Bone sialoprotein, integrin, bone morphogenic protein 2, PP2A (serine/threonine phosphatase), and c-fos. The total RNA was extracted from hMSCs cultured on the substrates (n = 6) at different time points (0, 7, 14, and 21 days) using the RNeasy Mini kit (Qiagen, Chatsworth, CA, USA). Following cDNA synthesis and qPCR, the relative gene expression was normalized to GAPDH and baseline expression. The P value was set at level

    Article Snippet: To quantify the levels of gene expression, the total RNA was extracted from hMSCs cultured on the substrates (n = 6) at different time points (0, 7, 14, and 21 days) using the RNeasy Mini kit (Qiagen, Chatsworth, CA, USA).

    Techniques: Expressing, Hemagglutination Assay, Cell Culture, Real-time Polymerase Chain Reaction

    Quantitative gene expression of PLLA/Col/HA at different time points during the differentiation process. (a) Gene expression of beta-catenin, frizzled, alkaline phosphatase, and osteopontin. (b) OC (BGALP), osteonectin, Runx2, and Col. (c) Bone sialoprotein, integrin, bone morphogenic protein 2, PP2A (serine/threonine phosphatase), and c-fos. The total RNA was extracted from hMSCs cultured on the substrates (n = 6) at different time points (0, 7, 14, and 21 days) using the RNeasy Mini kit (Qiagen, Chatsworth, CA, USA). Following cDNA synthesis and qPCR, the relative gene expression was normalized to GAPDH and baseline expression. The P value was set at level

    Journal: PLoS ONE

    Article Title: A Comparative Study on In Vitro Osteogenic Priming Potential of Electron Spun Scaffold PLLA/HA/Col, PLLA/HA, and PLLA/Col for Tissue Engineering Application

    doi: 10.1371/journal.pone.0104389

    Figure Lengend Snippet: Quantitative gene expression of PLLA/Col/HA at different time points during the differentiation process. (a) Gene expression of beta-catenin, frizzled, alkaline phosphatase, and osteopontin. (b) OC (BGALP), osteonectin, Runx2, and Col. (c) Bone sialoprotein, integrin, bone morphogenic protein 2, PP2A (serine/threonine phosphatase), and c-fos. The total RNA was extracted from hMSCs cultured on the substrates (n = 6) at different time points (0, 7, 14, and 21 days) using the RNeasy Mini kit (Qiagen, Chatsworth, CA, USA). Following cDNA synthesis and qPCR, the relative gene expression was normalized to GAPDH and baseline expression. The P value was set at level

    Article Snippet: To quantify the levels of gene expression, the total RNA was extracted from hMSCs cultured on the substrates (n = 6) at different time points (0, 7, 14, and 21 days) using the RNeasy Mini kit (Qiagen, Chatsworth, CA, USA).

    Techniques: Expressing, Hemagglutination Assay, Cell Culture, Real-time Polymerase Chain Reaction

    Quantitative gene expression of PLLA/Col at different time points during differentiation process. (a) Gene expression of beta-catenin, frizzled, alkaline phosphatase and osteopontin. (b) OC (BGALP), osteonectin, Runx2, and Col. (c) Bone sialoprotein, integrin, bone morphogenic protein 2, PP2A (serine/threonine phosphatase), and c-fos. The total RNA was extracted from hMSCs cultured on the substrates (n = 6) at different time points (0, 7, 14, and 21 days) using the RNeasy Mini kit (Qiagen, Chatsworth, CA, USA). Following cDNA synthesis and qPCR, the relative gene expression was normalized to GAPDH and baseline expression. The P value was set at level

    Journal: PLoS ONE

    Article Title: A Comparative Study on In Vitro Osteogenic Priming Potential of Electron Spun Scaffold PLLA/HA/Col, PLLA/HA, and PLLA/Col for Tissue Engineering Application

    doi: 10.1371/journal.pone.0104389

    Figure Lengend Snippet: Quantitative gene expression of PLLA/Col at different time points during differentiation process. (a) Gene expression of beta-catenin, frizzled, alkaline phosphatase and osteopontin. (b) OC (BGALP), osteonectin, Runx2, and Col. (c) Bone sialoprotein, integrin, bone morphogenic protein 2, PP2A (serine/threonine phosphatase), and c-fos. The total RNA was extracted from hMSCs cultured on the substrates (n = 6) at different time points (0, 7, 14, and 21 days) using the RNeasy Mini kit (Qiagen, Chatsworth, CA, USA). Following cDNA synthesis and qPCR, the relative gene expression was normalized to GAPDH and baseline expression. The P value was set at level

    Article Snippet: To quantify the levels of gene expression, the total RNA was extracted from hMSCs cultured on the substrates (n = 6) at different time points (0, 7, 14, and 21 days) using the RNeasy Mini kit (Qiagen, Chatsworth, CA, USA).

    Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    Comparison of RNA extracted from RSV infected (black bars) and uninfected (gray bars) HEp-2 cell lysates using the extraction cassette and RNeasy kit. Unextracted samples failed to report RSV RNA in either sample (mean ± s.d, n = 3).

    Journal: ACS applied materials & interfaces

    Article Title: Development of a low resource RNA extraction cassette based on surface tension valves

    doi: 10.1021/am2004009

    Figure Lengend Snippet: Comparison of RNA extracted from RSV infected (black bars) and uninfected (gray bars) HEp-2 cell lysates using the extraction cassette and RNeasy kit. Unextracted samples failed to report RSV RNA in either sample (mean ± s.d, n = 3).

    Article Snippet: The recovery efficiency of RNA extraction was compared to the RNeasy Mini kit (Qiagen, Germantown, MD), Dynabeads mRNA Direct kit (Invitrogen, Oslo, Norway) used according to manufacturer’s protocols, as well as the MagAttract RNA Cell Mini M48 kit (Qiagen, Germantown, MD) performed manually instead of with the Qiagen M48 BioRobot which was unavailable for these studies.

    Techniques: Infection

    The limit of detection of RNA detectable by RT-PCR after extraction from HEp-2 cell lysates spiked with known amounts of RSV RNA using either the continuous tubing extraction cassette (●) or the RNeasy kit (○) (mean ± s.d, n=3).

    Journal: ACS applied materials & interfaces

    Article Title: Development of a low resource RNA extraction cassette based on surface tension valves

    doi: 10.1021/am2004009

    Figure Lengend Snippet: The limit of detection of RNA detectable by RT-PCR after extraction from HEp-2 cell lysates spiked with known amounts of RSV RNA using either the continuous tubing extraction cassette (●) or the RNeasy kit (○) (mean ± s.d, n=3).

    Article Snippet: The recovery efficiency of RNA extraction was compared to the RNeasy Mini kit (Qiagen, Germantown, MD), Dynabeads mRNA Direct kit (Invitrogen, Oslo, Norway) used according to manufacturer’s protocols, as well as the MagAttract RNA Cell Mini M48 kit (Qiagen, Germantown, MD) performed manually instead of with the Qiagen M48 BioRobot which was unavailable for these studies.

    Techniques: Reverse Transcription Polymerase Chain Reaction

    The number of copies of RNA per μL extracted from RSV positive (black bars) and RSV negative (gray bars) nasal wash samples using five extraction methods were compared. Extractions were performed using prototype extraction cassette, RNeasy Mini

    Journal: ACS applied materials & interfaces

    Article Title: Development of a low resource RNA extraction cassette based on surface tension valves

    doi: 10.1021/am2004009

    Figure Lengend Snippet: The number of copies of RNA per μL extracted from RSV positive (black bars) and RSV negative (gray bars) nasal wash samples using five extraction methods were compared. Extractions were performed using prototype extraction cassette, RNeasy Mini

    Article Snippet: The recovery efficiency of RNA extraction was compared to the RNeasy Mini kit (Qiagen, Germantown, MD), Dynabeads mRNA Direct kit (Invitrogen, Oslo, Norway) used according to manufacturer’s protocols, as well as the MagAttract RNA Cell Mini M48 kit (Qiagen, Germantown, MD) performed manually instead of with the Qiagen M48 BioRobot which was unavailable for these studies.

    Techniques:

    Comparison of the percent of RSV RNA recovered after addition to TE buffer (A) or HEp-2 cell lysates (B) using the extraction cassette (left bars), RNeasy kit (middle bars), or no extraction (right bars) (mean ± s.d., n = 9). The recovery efficiency

    Journal: ACS applied materials & interfaces

    Article Title: Development of a low resource RNA extraction cassette based on surface tension valves

    doi: 10.1021/am2004009

    Figure Lengend Snippet: Comparison of the percent of RSV RNA recovered after addition to TE buffer (A) or HEp-2 cell lysates (B) using the extraction cassette (left bars), RNeasy kit (middle bars), or no extraction (right bars) (mean ± s.d., n = 9). The recovery efficiency

    Article Snippet: The recovery efficiency of RNA extraction was compared to the RNeasy Mini kit (Qiagen, Germantown, MD), Dynabeads mRNA Direct kit (Invitrogen, Oslo, Norway) used according to manufacturer’s protocols, as well as the MagAttract RNA Cell Mini M48 kit (Qiagen, Germantown, MD) performed manually instead of with the Qiagen M48 BioRobot which was unavailable for these studies.

    Techniques:

    A) Concentration of RNA extracted from HEK293 cells encapsulated in the four peptide hydrogels and cell-only controls using the three methods (see text for details). B Representative electrophoresis traces of total RNA extracted from cells encapsulated in the four peptide hydrogels and cell-only controls using the Tri Reagent and RNeasy MK methods and corresponding RIN values: cell-only controls (A+E), PGD-Alpha1 (B+F), PGD-Alpha2 (G), PGD-AlphaProB (C+H) and PGD-AlphaProC (D+I). C D) Representative UV spectra for RNA samples extracted using the TRI Reagent (C) and the RNeasy MK (D) methods from the four peptide hydrogels and cell-only control.

    Journal: PLoS ONE

    Article Title: RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

    doi: 10.1371/journal.pone.0197517

    Figure Lengend Snippet: A) Concentration of RNA extracted from HEK293 cells encapsulated in the four peptide hydrogels and cell-only controls using the three methods (see text for details). B Representative electrophoresis traces of total RNA extracted from cells encapsulated in the four peptide hydrogels and cell-only controls using the Tri Reagent and RNeasy MK methods and corresponding RIN values: cell-only controls (A+E), PGD-Alpha1 (B+F), PGD-Alpha2 (G), PGD-AlphaProB (C+H) and PGD-AlphaProC (D+I). C D) Representative UV spectra for RNA samples extracted using the TRI Reagent (C) and the RNeasy MK (D) methods from the four peptide hydrogels and cell-only control.

    Article Snippet: RNA was extracted using the following commercial kits: RNeasy Mini Kit® (Qiagen, Manchester, UK) or TRI Reagent® (Sigma-Aldrich, Dorset, UK).

    Techniques: Concentration Assay, Electrophoresis

    Ct values obtained for RT-qPCR performed using RNA extracted from the four peptide hydrogels as a template. RNA isolated from cells in suspension was used as a control. The RNA extracted using either the TRI Reagent method (A-B) or RNeasy MK method (C+D) was used as templates for the amplification of two housekeeping genes: GAPDH (A+C) and RPL13A (B+D). The cycle threshold (Ct) value was determined for three independent samples measured in triplicate (or two independent samples for PGD-Alpha1 using the RNeasy method). Data is shown as mean ± SEM. The mean values were compared to the non-encapsulated cell controls using a t-test; *, P ≤ 0.05.

    Journal: PLoS ONE

    Article Title: RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

    doi: 10.1371/journal.pone.0197517

    Figure Lengend Snippet: Ct values obtained for RT-qPCR performed using RNA extracted from the four peptide hydrogels as a template. RNA isolated from cells in suspension was used as a control. The RNA extracted using either the TRI Reagent method (A-B) or RNeasy MK method (C+D) was used as templates for the amplification of two housekeeping genes: GAPDH (A+C) and RPL13A (B+D). The cycle threshold (Ct) value was determined for three independent samples measured in triplicate (or two independent samples for PGD-Alpha1 using the RNeasy method). Data is shown as mean ± SEM. The mean values were compared to the non-encapsulated cell controls using a t-test; *, P ≤ 0.05.

    Article Snippet: RNA was extracted using the following commercial kits: RNeasy Mini Kit® (Qiagen, Manchester, UK) or TRI Reagent® (Sigma-Aldrich, Dorset, UK).

    Techniques: Quantitative RT-PCR, Isolation, Amplification

    Ct values obtained for RT-qPCR performed using RNA extracted using RNeasy MK method from the four peptide hydrogels pre-treated with pronase enzyme solution and the cell-only control as a template. RNA extracted was used as templates for the amplification of five housekeeping genes: GAPDH (A), RPL13A (B), ACTB (C), B2M (D) and RRN18s (E) commonly expressed in HEK293 cells. The cycle threshold (Ct) values are presented as the mean ± SEM for three independent samples measured in triplicate. *, P

    Journal: PLoS ONE

    Article Title: RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

    doi: 10.1371/journal.pone.0197517

    Figure Lengend Snippet: Ct values obtained for RT-qPCR performed using RNA extracted using RNeasy MK method from the four peptide hydrogels pre-treated with pronase enzyme solution and the cell-only control as a template. RNA extracted was used as templates for the amplification of five housekeeping genes: GAPDH (A), RPL13A (B), ACTB (C), B2M (D) and RRN18s (E) commonly expressed in HEK293 cells. The cycle threshold (Ct) values are presented as the mean ± SEM for three independent samples measured in triplicate. *, P

    Article Snippet: RNA was extracted using the following commercial kits: RNeasy Mini Kit® (Qiagen, Manchester, UK) or TRI Reagent® (Sigma-Aldrich, Dorset, UK).

    Techniques: Quantitative RT-PCR, Amplification

    Ferroportin is expressed in hPASMCs and regulated by hepcidin. Confluent hPASMCs were ( A ) either mock-treated or treated with 1 µg/mL hepcidin for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human Fpn (ferroportin) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) mock-treated for 24 h, cells lysed and total protein extracted, quantified by Bradford assay and 40 µg of protein separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blotting was performed using Rabbit anti-Fpn as primary and Goat anti-rabbit IgG conjugated with horse-radish peroxidase as secondary antibodies. Human intestinal lysates (Abcam) were used as positive control. N = 3 ( C ) either mock-treated or treated with 1 µg/mL hepcidin for 24 h, cells lysed and total protein extracted. Fpn expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 1 µg/mL hepcidin for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm; N = 5. Student’s t test was performed; **p

    Journal: Scientific Reports

    Article Title: The Hepcidin/Ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells

    doi: 10.1038/s41598-018-31095-0

    Figure Lengend Snippet: Ferroportin is expressed in hPASMCs and regulated by hepcidin. Confluent hPASMCs were ( A ) either mock-treated or treated with 1 µg/mL hepcidin for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human Fpn (ferroportin) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) mock-treated for 24 h, cells lysed and total protein extracted, quantified by Bradford assay and 40 µg of protein separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blotting was performed using Rabbit anti-Fpn as primary and Goat anti-rabbit IgG conjugated with horse-radish peroxidase as secondary antibodies. Human intestinal lysates (Abcam) were used as positive control. N = 3 ( C ) either mock-treated or treated with 1 µg/mL hepcidin for 24 h, cells lysed and total protein extracted. Fpn expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 1 µg/mL hepcidin for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm; N = 5. Student’s t test was performed; **p

    Article Snippet: Total RNA was extracted using the RNeasy Mini kit (Qiagen) following the manufacturer’s instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Bradford Assay, SDS Page, Western Blot, Positive Control, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Microscopy

    IL-6 increased hepcidin expression and down-regulated ferroportin in hPASMCs. Confluent hPASMCs were either mock-treated or treated with 10 ng/mL IL-6 ( A ) for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human hepcidin (Hamp-1) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) for 24 h, media supernatant collected and hepcidin secretion was quantitated using an ELISA kit (R D Systems). N = 4 ( C ) for 24 h cells lysed and total protein extracted. Ferroportin expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 10 ng/mL IL-6 for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm, N = 5. Student’s t test was performed; **p

    Journal: Scientific Reports

    Article Title: The Hepcidin/Ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells

    doi: 10.1038/s41598-018-31095-0

    Figure Lengend Snippet: IL-6 increased hepcidin expression and down-regulated ferroportin in hPASMCs. Confluent hPASMCs were either mock-treated or treated with 10 ng/mL IL-6 ( A ) for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human hepcidin (Hamp-1) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) for 24 h, media supernatant collected and hepcidin secretion was quantitated using an ELISA kit (R D Systems). N = 4 ( C ) for 24 h cells lysed and total protein extracted. Ferroportin expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 10 ng/mL IL-6 for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm, N = 5. Student’s t test was performed; **p

    Article Snippet: Total RNA was extracted using the RNeasy Mini kit (Qiagen) following the manufacturer’s instructions.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy

    hiPSCs transduced with the reporter vector containing miRNA targets indicate differentiation-specific reporter expression in neural lineages. EBs generated from pooled hiPSCs were differentiated into neural lineages using Noggin and SB431542. EBs were then transferred onto fibronectin coated 6-well plates and further differentiated in N2 medium. (A) Total RNA was isolated using QIAGEN's RNeasy Mini kit from the hiPSCs on day 0 (undifferentiated population: Undiff.) and day 30 (differentiated population: Diff.) after induction of differentiation. Total RNA (250 ng) was reverse-transcribed using QIAGEN's Omniscript reverse transcription kit and used as a template in subsequent PCR with 5-PRIME's HotMaster Taq DNA polymerase. PCR products were analyzed on a 2% agarose gel. GAPDH was used as an internal control. (B) Neural tube-like rosettes observed after the differentiation. (C) Dark pigmented melanocyte-like cells surrounding neural tube-like structures.

    Journal: PLoS ONE

    Article Title: Live Cell Monitoring of hiPSC Generation and Differentiation Using Differential Expression of Endogenous microRNAs

    doi: 10.1371/journal.pone.0011834

    Figure Lengend Snippet: hiPSCs transduced with the reporter vector containing miRNA targets indicate differentiation-specific reporter expression in neural lineages. EBs generated from pooled hiPSCs were differentiated into neural lineages using Noggin and SB431542. EBs were then transferred onto fibronectin coated 6-well plates and further differentiated in N2 medium. (A) Total RNA was isolated using QIAGEN's RNeasy Mini kit from the hiPSCs on day 0 (undifferentiated population: Undiff.) and day 30 (differentiated population: Diff.) after induction of differentiation. Total RNA (250 ng) was reverse-transcribed using QIAGEN's Omniscript reverse transcription kit and used as a template in subsequent PCR with 5-PRIME's HotMaster Taq DNA polymerase. PCR products were analyzed on a 2% agarose gel. GAPDH was used as an internal control. (B) Neural tube-like rosettes observed after the differentiation. (C) Dark pigmented melanocyte-like cells surrounding neural tube-like structures.

    Article Snippet: RNA extraction from hESC and hiPSC was performed using QIAGEN's RNeasy Mini kit following the manufacture's protocol (QIAGEN, Valencia, CA).

    Techniques: Transduction, Plasmid Preparation, Expressing, Generated, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Molecular characterization of hiPSCs transduced with the reporter vector containing miRNA targets. Total RNA was isolated using QIAGEN's RNeasy Mini kit from HFFs transduced with (4Fs/HFF) or without 4 reprogramming factors (HFF), hESCs (H1), and 4 different hiPSC clones transduced with (E/m#1, E/m#5, and E/m#101) or without (hiPSC#19) the reporter vector encoding EGFP miR-T/mCherry miR-T. Total RNA (250 ng) was reverse-transcribed using QIAGEN's Omniscript reverse transcription kit and used as a template in subsequent PCR with 5-PRIME's HotMaster Taq DNA polymerase. PCR products were analyzed on a 2% agarose gel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

    Journal: PLoS ONE

    Article Title: Live Cell Monitoring of hiPSC Generation and Differentiation Using Differential Expression of Endogenous microRNAs

    doi: 10.1371/journal.pone.0011834

    Figure Lengend Snippet: Molecular characterization of hiPSCs transduced with the reporter vector containing miRNA targets. Total RNA was isolated using QIAGEN's RNeasy Mini kit from HFFs transduced with (4Fs/HFF) or without 4 reprogramming factors (HFF), hESCs (H1), and 4 different hiPSC clones transduced with (E/m#1, E/m#5, and E/m#101) or without (hiPSC#19) the reporter vector encoding EGFP miR-T/mCherry miR-T. Total RNA (250 ng) was reverse-transcribed using QIAGEN's Omniscript reverse transcription kit and used as a template in subsequent PCR with 5-PRIME's HotMaster Taq DNA polymerase. PCR products were analyzed on a 2% agarose gel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

    Article Snippet: RNA extraction from hESC and hiPSC was performed using QIAGEN's RNeasy Mini kit following the manufacture's protocol (QIAGEN, Valencia, CA).

    Techniques: Transduction, Plasmid Preparation, Isolation, Clone Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Agilent 2100 Bioanalyzer electropherogram profiles of total RNA samples (HeLa cells) extracted with TRIzol reagent (green), MirVana kit (blue), and RNEasy kit (red). Inbox: magnification of small RNA profiles for the three samples (between 23 and 29 seconds).

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Quantification of Small Non-Coding RNAs Allows an Accurate Comparison of miRNA Expression Profiles

    doi: 10.1155/2009/659028

    Figure Lengend Snippet: Agilent 2100 Bioanalyzer electropherogram profiles of total RNA samples (HeLa cells) extracted with TRIzol reagent (green), MirVana kit (blue), and RNEasy kit (red). Inbox: magnification of small RNA profiles for the three samples (between 23 and 29 seconds).

    Article Snippet: In particular, TRIzol reagent allowed the highest LMW RNA recovery (22%–34% of total RNA), while RNEasy Mini Kit the lowest (2.5%–3%).

    Techniques:

    (a) Gel electrophoresis (polyacrylamide 15% stained with ethidium bromide) of LMW RNA samples (LCL) extracted with TRIzol reagent (lane 1), RNEasy kit (lane 2), and small RNA fraction enriched with MirVana kit (lane 3). LMW profile obtained with MirVana kit extraction is similar to that obtained with TRIzol reagent which is not shown for clarity. (b) Agilent 2100 Bioanalyzer electropherogram profile of LMW RNAs (LCL) extracted with TRIzol (black) superimposed on 5.8S (red), 5S (green), and tRNA (blue) bands eluted from polyacrylamide gel. (c) Lymphoblastoid (LCL) LMW RNA profile obtained after plotting the exported raw data from Agilent electropherogram ( ) together with the PeakFit fitted curve (solid line) and the component peak functions. Seven peaks below the LMW RNA profile were fitted by the software ( r 2 = 0.998693).

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Quantification of Small Non-Coding RNAs Allows an Accurate Comparison of miRNA Expression Profiles

    doi: 10.1155/2009/659028

    Figure Lengend Snippet: (a) Gel electrophoresis (polyacrylamide 15% stained with ethidium bromide) of LMW RNA samples (LCL) extracted with TRIzol reagent (lane 1), RNEasy kit (lane 2), and small RNA fraction enriched with MirVana kit (lane 3). LMW profile obtained with MirVana kit extraction is similar to that obtained with TRIzol reagent which is not shown for clarity. (b) Agilent 2100 Bioanalyzer electropherogram profile of LMW RNAs (LCL) extracted with TRIzol (black) superimposed on 5.8S (red), 5S (green), and tRNA (blue) bands eluted from polyacrylamide gel. (c) Lymphoblastoid (LCL) LMW RNA profile obtained after plotting the exported raw data from Agilent electropherogram ( ) together with the PeakFit fitted curve (solid line) and the component peak functions. Seven peaks below the LMW RNA profile were fitted by the software ( r 2 = 0.998693).

    Article Snippet: In particular, TRIzol reagent allowed the highest LMW RNA recovery (22%–34% of total RNA), while RNEasy Mini Kit the lowest (2.5%–3%).

    Techniques: Nucleic Acid Electrophoresis, Staining, Software

    Gel electrophoresis (agarose 1% stained with ethidium bromide) of RNA samples (from COS-1) extracted with TRIzol reagent (lane 1), MirVana kit (lane 2), RNEasy kit (lane 3), and LMW RNA fraction enriched with MirVana kit (lane 4).

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Quantification of Small Non-Coding RNAs Allows an Accurate Comparison of miRNA Expression Profiles

    doi: 10.1155/2009/659028

    Figure Lengend Snippet: Gel electrophoresis (agarose 1% stained with ethidium bromide) of RNA samples (from COS-1) extracted with TRIzol reagent (lane 1), MirVana kit (lane 2), RNEasy kit (lane 3), and LMW RNA fraction enriched with MirVana kit (lane 4).

    Article Snippet: In particular, TRIzol reagent allowed the highest LMW RNA recovery (22%–34% of total RNA), while RNEasy Mini Kit the lowest (2.5%–3%).

    Techniques: Nucleic Acid Electrophoresis, Staining

    Involvement of STAT5b in MSM-induced osteogenic marker genes in MSCs. (A) Bone marrow mesenchymal stem cells were cultured in the osteogenic medium at 5 days for ALP, 14 days for osteonectin (ON) and bone sialoprotein (BSP), and 21 days for osteocalcin (OCN) and osterix mRNA expression after the treatment with various concentrations (0, 10 and 20 mM) of MSM. RT-PCR was performed using the cDNA and primers for ALP, ON, BSP, OCN, osterix and 18S. Total RNA was isolated from the MSCs using an RNeasy kit. 18S was used as a control. (B) Bone marrow Mesenchymal stem cells and (C) C3H10T1/2 cells were cultured in osteogenic medium at 5 days for ALP and Runx2, 14 days for OPN and BSP, and 21 days for OCN and osterix mRNA expression after the treatment with 20 mM MSM. After culture, real-time PCR was performed. (D) Osteogenic differentiation marker genes (ALP, BSP, OCN, OPN, Osterix and Runx2) and STAT5b gene expression was analyzed at day 5, 14 and 21 after MSM treatment in C3H10T1/2 cells transfected with STAT5b siRNA or non-target siRNA. The effect of STAT5b knockdown on osteogenic marker genes was analyzed by real-time PCR. GAPDH was used as the internal control. The relative levels of mRNA were determined using densitometric analysis and normalized to the amount of GAPDH. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by t-test (*p

    Journal: PLoS ONE

    Article Title: MSM Enhances GH Signaling via the Jak2/STAT5b Pathway in Osteoblast-Like Cells and Osteoblast Differentiation through the Activation of STAT5b in MSCs

    doi: 10.1371/journal.pone.0047477

    Figure Lengend Snippet: Involvement of STAT5b in MSM-induced osteogenic marker genes in MSCs. (A) Bone marrow mesenchymal stem cells were cultured in the osteogenic medium at 5 days for ALP, 14 days for osteonectin (ON) and bone sialoprotein (BSP), and 21 days for osteocalcin (OCN) and osterix mRNA expression after the treatment with various concentrations (0, 10 and 20 mM) of MSM. RT-PCR was performed using the cDNA and primers for ALP, ON, BSP, OCN, osterix and 18S. Total RNA was isolated from the MSCs using an RNeasy kit. 18S was used as a control. (B) Bone marrow Mesenchymal stem cells and (C) C3H10T1/2 cells were cultured in osteogenic medium at 5 days for ALP and Runx2, 14 days for OPN and BSP, and 21 days for OCN and osterix mRNA expression after the treatment with 20 mM MSM. After culture, real-time PCR was performed. (D) Osteogenic differentiation marker genes (ALP, BSP, OCN, OPN, Osterix and Runx2) and STAT5b gene expression was analyzed at day 5, 14 and 21 after MSM treatment in C3H10T1/2 cells transfected with STAT5b siRNA or non-target siRNA. The effect of STAT5b knockdown on osteogenic marker genes was analyzed by real-time PCR. GAPDH was used as the internal control. The relative levels of mRNA were determined using densitometric analysis and normalized to the amount of GAPDH. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by t-test (*p

    Article Snippet: Total RNA was prepared using the RNeasy Mini kit (Qiagen) and cDNA was synthesized using the AccuPower RT PreMix kit (Bioneer) according to the manufacturer’s instructions.

    Techniques: Marker, Cell Culture, ALP Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction, Transfection

    Methylsulfonylmethane (MSM) activates the expression of growth hormone (GH) signaling-related mRNA in UMR 106 cells. Total RNA was isolated from the UMR-106 cells using an RNeasy kit. The cDNA was amplified using specific primers for insulin like growth factor-1 receptor (IGF-1R), the growth hormone receptor (GHR) or 18S. 18S was used as a control. (A) UMR-106 cells were treated with the indicated concentrations of MSM for 24 h. (B) The relative levels of IGF-1R and GHR mRNA were determined using densitometric analysis and normalized to the amount of 18S. (C) UMR-106 cells were left untreated or pretreated with 50 µM AG490 for 4 h then treated with MSM for 24 h. (D) The relative levels of IGF-1R and GHR mRNA were determined using densitometric analysis and normalized to the amount of 18S. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by ANOVA (***p

    Journal: PLoS ONE

    Article Title: MSM Enhances GH Signaling via the Jak2/STAT5b Pathway in Osteoblast-Like Cells and Osteoblast Differentiation through the Activation of STAT5b in MSCs

    doi: 10.1371/journal.pone.0047477

    Figure Lengend Snippet: Methylsulfonylmethane (MSM) activates the expression of growth hormone (GH) signaling-related mRNA in UMR 106 cells. Total RNA was isolated from the UMR-106 cells using an RNeasy kit. The cDNA was amplified using specific primers for insulin like growth factor-1 receptor (IGF-1R), the growth hormone receptor (GHR) or 18S. 18S was used as a control. (A) UMR-106 cells were treated with the indicated concentrations of MSM for 24 h. (B) The relative levels of IGF-1R and GHR mRNA were determined using densitometric analysis and normalized to the amount of 18S. (C) UMR-106 cells were left untreated or pretreated with 50 µM AG490 for 4 h then treated with MSM for 24 h. (D) The relative levels of IGF-1R and GHR mRNA were determined using densitometric analysis and normalized to the amount of 18S. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by ANOVA (***p

    Article Snippet: Total RNA was prepared using the RNeasy Mini kit (Qiagen) and cDNA was synthesized using the AccuPower RT PreMix kit (Bioneer) according to the manufacturer’s instructions.

    Techniques: Expressing, Isolation, Amplification

    The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.

    Journal: PLoS ONE

    Article Title: Filter paper-based spin column method for cost-efficient DNA or RNA purification

    doi: 10.1371/journal.pone.0203011

    Figure Lengend Snippet: The efficiency of filter paper for purification of nucleic acids from various sources using respective Qiagen kits. (A) Tomato genomic DNAs purified using Qiagen DNeasy plant mini kit. (B) Tomato total RNAs purified using Qiagen RNeasy plant mini kit. (C) PCR products of a GUS fragment purified using Qiagen QIAquick PCR purification kit. (D) PCR products of GUS fragment recovered from an agarose gel using a Qiagen QIAquick gel extraction kit. (E) pUC -19 plasmid DNAs purified using a Qiagen QIAprep spin miniprep kit. For each panel, from left to right are (Q) nucleic acid purified in experiments using original Qiagen spin column, (G) reassembled spin column using two layers of Whatman glass microfiber filters (Grade GF/F), and (P) reassembled spin column using two layers of Whatman qualitative filter paper, (Grade 3) respectively. Upper panel is quantification data based on three experimental replicates normalized according to performance of the Qiagen kit; lower panel is an image of agarose gel electrophoresis for the same volume of purified nucleic acids.

    Article Snippet: Plant total RNAs were purified using filter paper-based spin columns following the protocol of the Qiagen RNeasy Plant mini kit (RNeasy Mini Handbook.

    Techniques: Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Gel Extraction, Plasmid Preparation

    Evaluation of purification of tobacco genomic DNA and total RNA using filter paper-based spin columns with respective Qiagen kit buffers and homemade buffers. (A) Agarose gel electrophoresis for 2.5 μl tobacco genomic DNAs elution from purification experiments using Qiagen DNeasy plant mini kit buffers with Qiagen original spin column (Lane Q/Q), filter paper recharged used spin column (Lane Q/R) and filter paper-based homemade spin column (Lane Q/H*), followed by tobacco genomic DNAs purified using homemade buffer with Qiagen original spin column (Lane H/Q), filter paper recharged used spin column (Lane H/R) and filter paper-based homemade spin column (Lane H/H*). (B) UV spectrum curve of tobacco DNAs purified using Qiagen kit (Q/Q, black curve), filter paper recharged spin columns with Qiagen kit buffers (Q/R, blue curve) or homemade buffers (H/R, red curve) from the same amount leaf tissue. Y-axis is UV absorbance, and X-axis is wavelength (nM). (C) Amplification plots for three duplicated qPCR reactions contain 20 ng DNA purified using Qiagen kit (Q/Q, Blue curves) or DNA purified from filter paper recharged spin column with homemade buffer (H/R, Red curves) respectively. The x-axis is PCR cycle numbers, Y-axis is the level of SYBR fluorescence, and the green line is an arbitrary threshold to determine the Cq value (the fractional cycle number at which amplification curve meet threshold level). (D) MOPS-formaldehyde denaturing agarose gel electrophoresis separated 5 μl RNA purified using Qiagen RNeasy plant mini kit buffers with a Qiagen original spin column (Lane Q/Q), filter paper recharged used spin column (Lane Q/R) and homemade filter paper-based spin column (Lane Q/H*), followed total tobacco RNAs purified by using homemade buffer with Qiagen original spin column (Lane H/Q), filter paper recharged used spin column (Lane H/R) and filter paper-based homemade spin column (Lane H/H*). (E) UV spectrum of tobacco total RNA purified using Qiagen kit (Q/Q, black curve), filter paper recharged spin column with Qiagen RNeasy plant mini kit buffers (Q/R, blue curve) or homemade buffers (H/R, red curve). Y-axis is UV absorbance, and the X-axis is wavelength. (F) Amplification plots of three duplicated qRT-PCR reactions for 2.5 ng RNA purified using Qiagen kit (Q/Q, Blue curves) or RNA purified using filter paper recharged spin column with homemade buffer (H/R, Red curves) respectively. Note: * The starting material amount is 100 mg tobacco leaf tissue for experiments using a Qiagen spin column or filter paper recharged spin column, and half amount of plant sample (50 mg) used for homemade spin column purification. All DNAs or RNAs were eluted using 100 ul elution solution.

    Journal: PLoS ONE

    Article Title: Filter paper-based spin column method for cost-efficient DNA or RNA purification

    doi: 10.1371/journal.pone.0203011

    Figure Lengend Snippet: Evaluation of purification of tobacco genomic DNA and total RNA using filter paper-based spin columns with respective Qiagen kit buffers and homemade buffers. (A) Agarose gel electrophoresis for 2.5 μl tobacco genomic DNAs elution from purification experiments using Qiagen DNeasy plant mini kit buffers with Qiagen original spin column (Lane Q/Q), filter paper recharged used spin column (Lane Q/R) and filter paper-based homemade spin column (Lane Q/H*), followed by tobacco genomic DNAs purified using homemade buffer with Qiagen original spin column (Lane H/Q), filter paper recharged used spin column (Lane H/R) and filter paper-based homemade spin column (Lane H/H*). (B) UV spectrum curve of tobacco DNAs purified using Qiagen kit (Q/Q, black curve), filter paper recharged spin columns with Qiagen kit buffers (Q/R, blue curve) or homemade buffers (H/R, red curve) from the same amount leaf tissue. Y-axis is UV absorbance, and X-axis is wavelength (nM). (C) Amplification plots for three duplicated qPCR reactions contain 20 ng DNA purified using Qiagen kit (Q/Q, Blue curves) or DNA purified from filter paper recharged spin column with homemade buffer (H/R, Red curves) respectively. The x-axis is PCR cycle numbers, Y-axis is the level of SYBR fluorescence, and the green line is an arbitrary threshold to determine the Cq value (the fractional cycle number at which amplification curve meet threshold level). (D) MOPS-formaldehyde denaturing agarose gel electrophoresis separated 5 μl RNA purified using Qiagen RNeasy plant mini kit buffers with a Qiagen original spin column (Lane Q/Q), filter paper recharged used spin column (Lane Q/R) and homemade filter paper-based spin column (Lane Q/H*), followed total tobacco RNAs purified by using homemade buffer with Qiagen original spin column (Lane H/Q), filter paper recharged used spin column (Lane H/R) and filter paper-based homemade spin column (Lane H/H*). (E) UV spectrum of tobacco total RNA purified using Qiagen kit (Q/Q, black curve), filter paper recharged spin column with Qiagen RNeasy plant mini kit buffers (Q/R, blue curve) or homemade buffers (H/R, red curve). Y-axis is UV absorbance, and the X-axis is wavelength. (F) Amplification plots of three duplicated qRT-PCR reactions for 2.5 ng RNA purified using Qiagen kit (Q/Q, Blue curves) or RNA purified using filter paper recharged spin column with homemade buffer (H/R, Red curves) respectively. Note: * The starting material amount is 100 mg tobacco leaf tissue for experiments using a Qiagen spin column or filter paper recharged spin column, and half amount of plant sample (50 mg) used for homemade spin column purification. All DNAs or RNAs were eluted using 100 ul elution solution.

    Article Snippet: Plant total RNAs were purified using filter paper-based spin columns following the protocol of the Qiagen RNeasy Plant mini kit (RNeasy Mini Handbook.

    Techniques: Purification, Agarose Gel Electrophoresis, Amplification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Fluorescence, Quantitative RT-PCR

    Sorting small cell populations directly into the lysis buffer of the RNA isolation kit improves RNA quality and yield. ( a ) RQN values ( b ) and RNA yield of RNA samples isolated from a range of cell numbers (5000–200,000) when sorting directly into the lysis buffer of the RNA isolation kit or collecting the cells first into a collection medium. RQN values ( c ) and RNA yield ( d ) of RNA samples isolated from a range of cell numbers (5000–200,000) sorted directly into the lysis buffer or sorted into a collection buffer first. But here the volume of the lysis buffer was amended not to exceed its maximum dilution point caused by the sorting procedure. Left panels show samples isolated with the RNeasy plus micro kit, right panels with the RNAqueous micro kit. Average of two biological replicates is shown. Error bars indicate SEM

    Journal: BMC Genomics

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

    doi: 10.1186/s12864-019-5608-2

    Figure Lengend Snippet: Sorting small cell populations directly into the lysis buffer of the RNA isolation kit improves RNA quality and yield. ( a ) RQN values ( b ) and RNA yield of RNA samples isolated from a range of cell numbers (5000–200,000) when sorting directly into the lysis buffer of the RNA isolation kit or collecting the cells first into a collection medium. RQN values ( c ) and RNA yield ( d ) of RNA samples isolated from a range of cell numbers (5000–200,000) sorted directly into the lysis buffer or sorted into a collection buffer first. But here the volume of the lysis buffer was amended not to exceed its maximum dilution point caused by the sorting procedure. Left panels show samples isolated with the RNeasy plus micro kit, right panels with the RNAqueous micro kit. Average of two biological replicates is shown. Error bars indicate SEM

    Article Snippet: The RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen are specifically designed for purification of total RNA from a small amount of cells ( < 200,000 cells).

    Techniques: Lysis, Isolation

    Sorting cells directly into the lysis buffer preserves and protects RNA from degradation. RQN values of individual RNA samples isolated at specific time points after FACS sorting of 20,000 fli1a:EGFP cells. Cells were sorted directly in the lysis buffer of the kit or first captured into a collection medium. Left panel shows samples isolated with the RNAqueous micro kit, right panel with the RNeasy plus micro kit

    Journal: BMC Genomics

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

    doi: 10.1186/s12864-019-5608-2

    Figure Lengend Snippet: Sorting cells directly into the lysis buffer preserves and protects RNA from degradation. RQN values of individual RNA samples isolated at specific time points after FACS sorting of 20,000 fli1a:EGFP cells. Cells were sorted directly in the lysis buffer of the kit or first captured into a collection medium. Left panel shows samples isolated with the RNAqueous micro kit, right panel with the RNeasy plus micro kit

    Article Snippet: The RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen are specifically designed for purification of total RNA from a small amount of cells ( < 200,000 cells).

    Techniques: Lysis, Isolation, FACS

    Comparative qualitative analysis of RNA purified with the Rnaqeuous and Rneasy micro kit. ( a ) Boxplots showing RQN values and ( b ) RNA yield of 10 RNA samples purified from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent RQN values of individual samples. ( c ) Boxplots showing 5′-3′ delta-Cq (dCq) values calculated from a 5′ and a 3′ RT-qPCR assay of 7 RNA samples isolated from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent 5′-3′ dCq values of individual samples. * P

    Journal: BMC Genomics

    Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

    doi: 10.1186/s12864-019-5608-2

    Figure Lengend Snippet: Comparative qualitative analysis of RNA purified with the Rnaqeuous and Rneasy micro kit. ( a ) Boxplots showing RQN values and ( b ) RNA yield of 10 RNA samples purified from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent RQN values of individual samples. ( c ) Boxplots showing 5′-3′ delta-Cq (dCq) values calculated from a 5′ and a 3′ RT-qPCR assay of 7 RNA samples isolated from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent 5′-3′ dCq values of individual samples. * P

    Article Snippet: The RNAqueous Micro Total RNA Isolation Kit from Ambion and the RNeasy Plus Micro Kit from Qiagen are specifically designed for purification of total RNA from a small amount of cells ( < 200,000 cells).

    Techniques: Purification, FACS, Quantitative RT-PCR, Isolation

    Down-regulation of Dusp3 and Psme3 in A/J are responsible for increased NF-κB signaling activity. ( A ) The expression of Dcaf7 , Dusp3 , and Psme3 in A/J was significantly lower than C57BL/6J under both non-infected and S. aureus infected conditions. Eight-week-old male A/J and C57BL/6J mice (n = 6) were challenged (i.p.) by S. aureus Sanger 476 at 10 7 CFU/g or DPBS. At two hours post-infection whole blood RNAs was extracted by RNeasy followed by RT-PCR and qPCR. Dcaf7 (0.81 fold; p

    Journal: PLoS Pathogens

    Article Title: Dusp3 and Psme3 Are Associated with Murine Susceptibility to Staphylococcus aureus Infection and Human Sepsis

    doi: 10.1371/journal.ppat.1004149

    Figure Lengend Snippet: Down-regulation of Dusp3 and Psme3 in A/J are responsible for increased NF-κB signaling activity. ( A ) The expression of Dcaf7 , Dusp3 , and Psme3 in A/J was significantly lower than C57BL/6J under both non-infected and S. aureus infected conditions. Eight-week-old male A/J and C57BL/6J mice (n = 6) were challenged (i.p.) by S. aureus Sanger 476 at 10 7 CFU/g or DPBS. At two hours post-infection whole blood RNAs was extracted by RNeasy followed by RT-PCR and qPCR. Dcaf7 (0.81 fold; p

    Article Snippet: At two hours post infection blood RNA were extracted by QIAGEN RNeasy Protect Animal Blood Kit, followed by reverse-transcription PCR and SYBR-green quantitative-PCR.

    Techniques: Activity Assay, Expressing, Infection, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Quantitative-PCR confirmed elevation of cytokine production in macrophages transfected by Dusp3 and Psme3 siRNA or BMDMs from CSS11(GM-CSF, IL-1β, IL-6, and TNF-α). ( A ) Down-regulation of Dusp3 and Psme3 by siRNA led to increased cytokine RNA expression upon S. aureus challenge in RAW264.7 macrophages. At three hours post-infection total RNA was extracted followed by reverse-transcription PCR and SYBR-Green qPCR. The expression of all genes were normalized to 18s rRNA. The expression level of GM-CSF, IL-1β, IL-6, and TNF-α was higher in Dusp3 knockdown RAW cells, and the level of GM-CSF and IL-6 was higher in Psme3 knockdown RAW cells. p-value smaller than 0.05 was considered significant. ( B ) BMDMs cytokine RNA production in CSS11 mice was significantly higher than in C57BL/6J upon S. aureus infection. 2×10 6 BMDMs were seeded to single wells in a 6-well plate the day before infection. At three hours post-infection, RNA was extracted using RNeasy followed by RT-PCR and qPCR. The expression levels of GM-CSF, IL-1β, IL-6, and TNF-α were significantly higher in BMDMs from CSS11 mice. The expression of all genes were normalized to 18s rRNA. p-value smaller than 0.05 was considered significant. ( C ) Down-regulation of Dusp3 and Psme3 by siRNA led to increased cytokine RNA expression upon S. aureus challenge in BMDMs of C57BL/6J. The expression level of IL-6 was higher in Dusp3 siRNA transfected BMDMs, and the expression of TNF-α was higher in both Dusp3 and Psme3 siRNA transfected BMDMs.

    Journal: PLoS Pathogens

    Article Title: Dusp3 and Psme3 Are Associated with Murine Susceptibility to Staphylococcus aureus Infection and Human Sepsis

    doi: 10.1371/journal.ppat.1004149

    Figure Lengend Snippet: Quantitative-PCR confirmed elevation of cytokine production in macrophages transfected by Dusp3 and Psme3 siRNA or BMDMs from CSS11(GM-CSF, IL-1β, IL-6, and TNF-α). ( A ) Down-regulation of Dusp3 and Psme3 by siRNA led to increased cytokine RNA expression upon S. aureus challenge in RAW264.7 macrophages. At three hours post-infection total RNA was extracted followed by reverse-transcription PCR and SYBR-Green qPCR. The expression of all genes were normalized to 18s rRNA. The expression level of GM-CSF, IL-1β, IL-6, and TNF-α was higher in Dusp3 knockdown RAW cells, and the level of GM-CSF and IL-6 was higher in Psme3 knockdown RAW cells. p-value smaller than 0.05 was considered significant. ( B ) BMDMs cytokine RNA production in CSS11 mice was significantly higher than in C57BL/6J upon S. aureus infection. 2×10 6 BMDMs were seeded to single wells in a 6-well plate the day before infection. At three hours post-infection, RNA was extracted using RNeasy followed by RT-PCR and qPCR. The expression levels of GM-CSF, IL-1β, IL-6, and TNF-α were significantly higher in BMDMs from CSS11 mice. The expression of all genes were normalized to 18s rRNA. p-value smaller than 0.05 was considered significant. ( C ) Down-regulation of Dusp3 and Psme3 by siRNA led to increased cytokine RNA expression upon S. aureus challenge in BMDMs of C57BL/6J. The expression level of IL-6 was higher in Dusp3 siRNA transfected BMDMs, and the expression of TNF-α was higher in both Dusp3 and Psme3 siRNA transfected BMDMs.

    Article Snippet: At two hours post infection blood RNA were extracted by QIAGEN RNeasy Protect Animal Blood Kit, followed by reverse-transcription PCR and SYBR-green quantitative-PCR.

    Techniques: Real-time Polymerase Chain Reaction, Transfection, RNA Expression, Infection, Polymerase Chain Reaction, SYBR Green Assay, Expressing, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    2′- O methylation of internal adenosine. (A) Incorporation of 3 H-methyl into polyA. Homopolymer RNAs (1 µg) were incubated with 2 µg of DENV-4 MTase in the presence of [ 3 H-methyl]-SAM. After the methylation reaction, the un-incorporated [ 3 H-methyl]-SAM was removed by RNeasy kit. The amount of 3 H-methyl incorporation was measured by a MicroBeta counting. (B) SPA-based methylation analysis of oligo (A) 12 , (Am) 12 , and (m 6 ,m 6 A) 12 . All three RNA oligos were 3′-end biotinylated to facilitate SPA analysis. Am indicates that the 2′-OH of adenosine is methylated. m 6 ,m 6 A indicates that the amino N 6 position of adenosine is double methylated. (C) SPA-based methylation analysis of DENV-1 RNA. pppA-RNAs, representing the 5′ 211 nt of DENV-1 genome, were in vitro transcribed using biotinylated-CTP plus unmodified ATP, 2′- O -methyladenosine triphosphate (AmTP), or N 6 methyl adenosine triphosphate (m 6 ATP). The transcription reactions generated pppA-RNA, ppp(Am)-RNA, and ppp(m 6 A)RNA, respectively. The RNAs were then subjected to SPA-based internal methylation analysis. Average results and standard deviations from three independent experiments are presented.

    Journal: PLoS Pathogens

    Article Title: 2?-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

    doi: 10.1371/journal.ppat.1002642

    Figure Lengend Snippet: 2′- O methylation of internal adenosine. (A) Incorporation of 3 H-methyl into polyA. Homopolymer RNAs (1 µg) were incubated with 2 µg of DENV-4 MTase in the presence of [ 3 H-methyl]-SAM. After the methylation reaction, the un-incorporated [ 3 H-methyl]-SAM was removed by RNeasy kit. The amount of 3 H-methyl incorporation was measured by a MicroBeta counting. (B) SPA-based methylation analysis of oligo (A) 12 , (Am) 12 , and (m 6 ,m 6 A) 12 . All three RNA oligos were 3′-end biotinylated to facilitate SPA analysis. Am indicates that the 2′-OH of adenosine is methylated. m 6 ,m 6 A indicates that the amino N 6 position of adenosine is double methylated. (C) SPA-based methylation analysis of DENV-1 RNA. pppA-RNAs, representing the 5′ 211 nt of DENV-1 genome, were in vitro transcribed using biotinylated-CTP plus unmodified ATP, 2′- O -methyladenosine triphosphate (AmTP), or N 6 methyl adenosine triphosphate (m 6 ATP). The transcription reactions generated pppA-RNA, ppp(Am)-RNA, and ppp(m 6 A)RNA, respectively. The RNAs were then subjected to SPA-based internal methylation analysis. Average results and standard deviations from three independent experiments are presented.

    Article Snippet: After incubation at room temperature for 1 h, the unincorporated [3 H]-SAM was removed by RNeasy kit (Qiagen, Valencia, CA USA) according to the manufacture's instruction.

    Techniques: Methylation, Incubation, In Vitro, Generated

    Comparison of internal methylation efficiencies between DENV RNAs and host ribosomal RNAs. (A) Full-length (FL) and 3′ truncated RNAs of DENV-1. pppAG-RNAs, representing the FL and a set of 3′ terminally truncated DENV-1 RNAs, were in vitro synthesized. Numbers indicate nucleoside positions of DENV-1 genome (GenBank accession number U88535). (B) Internal methylation analysis. An equal mass (0.5 µg) of FL and truncated DENV-1 RNAs, and human ribosomal 18 S and 28 S RNAs was treated with DENV MTase in the presence of [ 3 H-methyl]-SAM. The reactions were purified through an RNeasy column to remove unincorporated [ 3 H-methyl]-SAM. The purified RNAs were quantified for internal methylation by a MicroBeta counter. Average results from three experiments are shown; error bars represent standard deviations.

    Journal: PLoS Pathogens

    Article Title: 2?-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

    doi: 10.1371/journal.ppat.1002642

    Figure Lengend Snippet: Comparison of internal methylation efficiencies between DENV RNAs and host ribosomal RNAs. (A) Full-length (FL) and 3′ truncated RNAs of DENV-1. pppAG-RNAs, representing the FL and a set of 3′ terminally truncated DENV-1 RNAs, were in vitro synthesized. Numbers indicate nucleoside positions of DENV-1 genome (GenBank accession number U88535). (B) Internal methylation analysis. An equal mass (0.5 µg) of FL and truncated DENV-1 RNAs, and human ribosomal 18 S and 28 S RNAs was treated with DENV MTase in the presence of [ 3 H-methyl]-SAM. The reactions were purified through an RNeasy column to remove unincorporated [ 3 H-methyl]-SAM. The purified RNAs were quantified for internal methylation by a MicroBeta counter. Average results from three experiments are shown; error bars represent standard deviations.

    Article Snippet: After incubation at room temperature for 1 h, the unincorporated [3 H]-SAM was removed by RNeasy kit (Qiagen, Valencia, CA USA) according to the manufacture's instruction.

    Techniques: Methylation, In Vitro, Synthesized, Purification