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  • 90
    Thermo Fisher genejet pcr purification kit
    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and <t>PCR</t> Clean-Up System (Promega); Th, <t>GeneJET</t> PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
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    Fisher Scientific genejet pcr purification kit
    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and <t>PCR</t> Clean-Up System (Promega); Th, <t>GeneJET</t> PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
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    Thermo Fisher genejet gel extraction kit
    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and <t>PCR</t> Clean-Up System (Promega); Th, <t>GeneJET</t> PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
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    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and <t>PCR</t> Clean-Up System (Promega); Th, <t>GeneJET</t> PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown
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    ) was digested with 2 U of TthHB27I in REase buffer supplemented with 100 μM of the selected effector and 25% (v/v) DMSO at 65 °C. The pictures on the right side of the figure’s panels show the theoretical arrangement of the <t>DNA</t> bands in an agarose gel after digesting the given substrate DNA with TthHB27I under standard conditions. a Cleavage pattern of non-methylated 1789 bp PCR fragment DNA. Lane M1, <t>GeneRuler</t> 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder, lane K, untreated DNA; lane 1, cleavage reaction in the absence of effector and DMSO; lane 2, in the presence of DMSO only; lane 3, in the presence of SAM only; lane 4, in the presence of SAM and DMSO; lane 5, in the presence of SIN only; lane 6, in the presence of SIN and DMSO. b The same as ( a ), but with previously methylated 1789 bp PCR fragment. c The same as ( a ), but with 1850 bp PCR fragment without TthHB27I cognate recognition sequence
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    MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression ( A ) or downregulation ( B ) was then checked by <t>qRT-PCR</t> and compared to that of their respective controls in MCF7 cells. ( C , D ) MED1 was overexpressed ( C ) or inhibited ( D ) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small <t>RNA.</t> The graphical data points represent mean + S.D of at least three independent experiments (**P
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    MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression ( A ) or downregulation ( B ) was then checked by <t>qRT-PCR</t> and compared to that of their respective controls in MCF7 cells. ( C , D ) MED1 was overexpressed ( C ) or inhibited ( D ) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small <t>RNA.</t> The graphical data points represent mean + S.D of at least three independent experiments (**P
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    MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression ( A ) or downregulation ( B ) was then checked by <t>qRT-PCR</t> and compared to that of their respective controls in MCF7 cells. ( C , D ) MED1 was overexpressed ( C ) or inhibited ( D ) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small <t>RNA.</t> The graphical data points represent mean + S.D of at least three independent experiments (**P
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    MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression ( A ) or downregulation ( B ) was then checked by <t>qRT-PCR</t> and compared to that of their respective controls in MCF7 cells. ( C , D ) MED1 was overexpressed ( C ) or inhibited ( D ) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small <t>RNA.</t> The graphical data points represent mean + S.D of at least three independent experiments (**P
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    MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression ( A ) or downregulation ( B ) was then checked by <t>qRT-PCR</t> and compared to that of their respective controls in MCF7 cells. ( C , D ) MED1 was overexpressed ( C ) or inhibited ( D ) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small <t>RNA.</t> The graphical data points represent mean + S.D of at least three independent experiments (**P
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    MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression ( A ) or downregulation ( B ) was then checked by <t>qRT-PCR</t> and compared to that of their respective controls in MCF7 cells. ( C , D ) MED1 was overexpressed ( C ) or inhibited ( D ) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small <t>RNA.</t> The graphical data points represent mean + S.D of at least three independent experiments (**P
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    MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression ( A ) or downregulation ( B ) was then checked by <t>qRT-PCR</t> and compared to that of their respective controls in MCF7 cells. ( C , D ) MED1 was overexpressed ( C ) or inhibited ( D ) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small <t>RNA.</t> The graphical data points represent mean + S.D of at least three independent experiments (**P
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    MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression ( A ) or downregulation ( B ) was then checked by <t>qRT-PCR</t> and compared to that of their respective controls in MCF7 cells. ( C , D ) MED1 was overexpressed ( C ) or inhibited ( D ) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small <t>RNA.</t> The graphical data points represent mean + S.D of at least three independent experiments (**P
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    MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression ( A ) or downregulation ( B ) was then checked by <t>qRT-PCR</t> and compared to that of their respective controls in MCF7 cells. ( C , D ) MED1 was overexpressed ( C ) or inhibited ( D ) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small <t>RNA.</t> The graphical data points represent mean + S.D of at least three independent experiments (**P
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    MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression ( A ) or downregulation ( B ) was then checked by <t>qRT-PCR</t> and compared to that of their respective controls in MCF7 cells. ( C , D ) MED1 was overexpressed ( C ) or inhibited ( D ) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small <t>RNA.</t> The graphical data points represent mean + S.D of at least three independent experiments (**P
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    MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression ( A ) or downregulation ( B ) was then checked by <t>qRT-PCR</t> and compared to that of their respective controls in MCF7 cells. ( C , D ) MED1 was overexpressed ( C ) or inhibited ( D ) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small <t>RNA.</t> The graphical data points represent mean + S.D of at least three independent experiments (**P
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    Efficiency of DNA removal by DNaseI and its fusion variants during <t>RNA</t> purification procedure. DNaseDT denotes the fusion with the (HhH) 2 domain of DNase from an extremely salt tolerant bacterium Thioalkalivibrio sp. K90mix , DNaseBS denotes the fusion with homologous domain of ComEA protein from Bacillus subtilis . The upper picture represents quantitative evaluation of undigested DNA remaining in eluates (RT-qPCR without added reverse transcriptase). The lower picture represents RT-qPCR results obtained using the same eluates. In the both pictures labels on the horizontal axis indicate the used nuclease, amount of it and the dilution ratio of the eluates before reverse transcription step and quantitation of DNA. A modified protocol of <t>GeneJET™</t> Whole Blood RNA Purification Mini Kit (Thermo Fisher Scientific, #K0761) was followed and RNA purification columns supplied by the manufacturer were used. During the experiment we have purified total blood RNA. Three arbitrary blood samples were analysed. DNA digestion was performed directly on a filter of a RNA purification column, were respective DNase enzyme was loaded.
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    DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Journal: BMC Genomics

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application

    doi: 10.1186/s12864-017-4371-5

    Figure Lengend Snippet: DNA purification reagents vary in their ability to recover low amounts of DNA from de-crosslinked chromatin. a Recovered DNA amount by different DNA purification reagents from de-crosslinked chromatin. De-crosslinked chromatin estimated to include 1 ng range DNA in ChIP elution buffer was purified following the manufacturer’s instructions. The data were generated from triplicate DNA samples derived from three independent preparations. Zy, ChIP DNA Clean Concentrator™ (Zymo Research); Pr, Wizard® SV Gel and PCR Clean-Up System (Promega); Th, GeneJET PCR Purification Kit (Thermo Fisher Scientific); In, PureLink® PCR Purification Kit (Invitrogen); Ne, Monarch® PCR DNA Cleanup Kit (New England Biolabs); Am, Chromatin IP DNA Purification Kit (Active Motif); Qp, QIAquick PCR Purification Kit (Qiagen); Qm, MinElute PCR Purification Kit (Qiagen); Ba, Agencourt AMPure XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); Br, RNAClean™ XP kit (Beckman, chromatin to beads ratio from 1:1.25 to 1:2); PC, phenol/chloroform extraction. b Interference of PCR amplification by purified eluent of purification reagents. 9 μL eluent was mixed with 1 μL 166 bp of Drosophila probe DNA (0.0001 ng), and the resulting mixture was used as the template in 20 μl of real-time PCR reaction. The Ct value for Drosophila probe DNA from TE buffer was set as 100%. The experiment was repeated 3 times using de-crosslinked chromatin estimated to include 1 ng of DNA. c Size profiles of DNA purified by different reagents. The DNAs purified from de-crosslinked chromatin estimated to include 50 ng range DNA was analyzed by AATI Fragment Analyzer. DNA size (bp) is shown

    Article Snippet: Similarly, DNAs were purified from de-crosslinked chromatin estimated to include 1 ng, 5 ng, 10 ng, and 50 ng of DNA after treatment of RNase A and proteinase K. The following reagents were used in the experiment: ChIP DNA Clean & Concentrator™ (Cat. # D5205) from Zymo Research (Zy) (Irvine, CA); Wizard® SV Gel and PCR Clean-Up System (Cat. # A9281) from Promega (Pr) (Fitchburg, WI); GeneJET PCR Purification Kit (Cat. # K0701) from Thermo Fisher Scientific (Th) (Waltham, MA); PureLink® PCR Purification Kit (Cat. # K310001) from Invitrogen (In) (Carlsbad, CA); Monarch® PCR & DNA Cleanup Kit (Cat. # T1030S) from New England Biolabs (Ne) (Ipswich, MA); Chromatin IP DNA Purification Kit (Cat. # 58002) from Active Motif (Am) (Carlsbad, CA); QIAquick PCR Purification Kit (Cat. # 28106) from Qiagen (Qp) (Valencia, CA), MinElute PCR Purification Kit (Cat. # 28006) from Qiagen (Qm); Agencourt AMPure XP (Cat. # A63881) from Beckman (Ba) (Indianapolis, IN), RNAClean™ XP (Cat. # A63987) from Beckman (Br), and phenol/chloroform extraction (PC) (Additional file ).

    Techniques: DNA Purification, Chromatin Immunoprecipitation, Purification, Generated, Derivative Assay, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    ) was digested with 2 U of TthHB27I in REase buffer supplemented with 100 μM of the selected effector and 25% (v/v) DMSO at 65 °C. The pictures on the right side of the figure’s panels show the theoretical arrangement of the DNA bands in an agarose gel after digesting the given substrate DNA with TthHB27I under standard conditions. a Cleavage pattern of non-methylated 1789 bp PCR fragment DNA. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder, lane K, untreated DNA; lane 1, cleavage reaction in the absence of effector and DMSO; lane 2, in the presence of DMSO only; lane 3, in the presence of SAM only; lane 4, in the presence of SAM and DMSO; lane 5, in the presence of SIN only; lane 6, in the presence of SIN and DMSO. b The same as ( a ), but with previously methylated 1789 bp PCR fragment. c The same as ( a ), but with 1850 bp PCR fragment without TthHB27I cognate recognition sequence

    Journal: BMC Genomics

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

    doi: 10.1186/s12864-018-4748-0

    Figure Lengend Snippet: ) was digested with 2 U of TthHB27I in REase buffer supplemented with 100 μM of the selected effector and 25% (v/v) DMSO at 65 °C. The pictures on the right side of the figure’s panels show the theoretical arrangement of the DNA bands in an agarose gel after digesting the given substrate DNA with TthHB27I under standard conditions. a Cleavage pattern of non-methylated 1789 bp PCR fragment DNA. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder, lane K, untreated DNA; lane 1, cleavage reaction in the absence of effector and DMSO; lane 2, in the presence of DMSO only; lane 3, in the presence of SAM only; lane 4, in the presence of SAM and DMSO; lane 5, in the presence of SIN only; lane 6, in the presence of SIN and DMSO. b The same as ( a ), but with previously methylated 1789 bp PCR fragment. c The same as ( a ), but with 1850 bp PCR fragment without TthHB27I cognate recognition sequence

    Article Snippet: DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania).

    Techniques: Agarose Gel Electrophoresis, Methylation, Polymerase Chain Reaction, Sequencing

    Cleavage of λ DNA under TthHB27I specificity relaxation conditions. a Cleavage pattern of λ DNA digested with TthHB27I under various conditions. 0.5 μg of DNA substrate was digested with 2 U of TthHB27I in REase buffer supplemented with 100 μM SAM and/or 25% (v/v) DMSO for 4 h at 65 °C. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, digestion in the presence of SAM; lane 2, digestion in the presence of DMSO; lane 3, digestion in the presence of both SAM and DMSO. b The same as ( a ), but SIN was used instead of SAM. c Distribution of insert lengths in 200 clones randomly selected from TthHB27I/DMSO/SAM-generated λ DNA library. d The same as ( c ), but for TthHB27I/DMSO/SIN-generated λ DNA library

    Journal: BMC Genomics

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

    doi: 10.1186/s12864-018-4748-0

    Figure Lengend Snippet: Cleavage of λ DNA under TthHB27I specificity relaxation conditions. a Cleavage pattern of λ DNA digested with TthHB27I under various conditions. 0.5 μg of DNA substrate was digested with 2 U of TthHB27I in REase buffer supplemented with 100 μM SAM and/or 25% (v/v) DMSO for 4 h at 65 °C. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, digestion in the presence of SAM; lane 2, digestion in the presence of DMSO; lane 3, digestion in the presence of both SAM and DMSO. b The same as ( a ), but SIN was used instead of SAM. c Distribution of insert lengths in 200 clones randomly selected from TthHB27I/DMSO/SAM-generated λ DNA library. d The same as ( c ), but for TthHB27I/DMSO/SIN-generated λ DNA library

    Article Snippet: DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania).

    Techniques: Clone Assay, Generated

    ) was incubated with 2 U of TthHB27I for 1 h at 65 °C in REase buffer with increasing amount of DMSO. Reaction mixtures were precipitated and electrophoresed in 1.3% agarose/TBE gels. The pictures on the right side of the figure show the theoretical arrangement of the DNA bands in an agarose gel after digesting the substrate DNA with TthHB27I under standard conditions. a Digestions performed only with the addition of DMSO, without any cofactor. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested PCR fragment; lane 1, 0% ( v /v) DMSO present in the reaction mixture; lane 2, with 5% (v/v) DMSO; lane 3, with 10% (v/v) DMSO; lane 4, with 15% (v/v) DMSO; lane 5, with 20% (v/v) DMSO; lane 6, with 25% (v/v) DMSO; lane 7, with 30% (v/v) DMSO. As in ( a ), but in the presence of 100 μM SAM ( b ); 100 μM SIN ( c ); 100 μM SAC ( d ); 100 μM SAH ( e ); 100 μM ATP ( f )

    Journal: BMC Genomics

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

    doi: 10.1186/s12864-018-4748-0

    Figure Lengend Snippet: ) was incubated with 2 U of TthHB27I for 1 h at 65 °C in REase buffer with increasing amount of DMSO. Reaction mixtures were precipitated and electrophoresed in 1.3% agarose/TBE gels. The pictures on the right side of the figure show the theoretical arrangement of the DNA bands in an agarose gel after digesting the substrate DNA with TthHB27I under standard conditions. a Digestions performed only with the addition of DMSO, without any cofactor. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested PCR fragment; lane 1, 0% ( v /v) DMSO present in the reaction mixture; lane 2, with 5% (v/v) DMSO; lane 3, with 10% (v/v) DMSO; lane 4, with 15% (v/v) DMSO; lane 5, with 20% (v/v) DMSO; lane 6, with 25% (v/v) DMSO; lane 7, with 30% (v/v) DMSO. As in ( a ), but in the presence of 100 μM SAM ( b ); 100 μM SIN ( c ); 100 μM SAC ( d ); 100 μM SAH ( e ); 100 μM ATP ( f )

    Article Snippet: DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania).

    Techniques: Incubation, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    Controlling of partial cleavage of genomic DNAs by TthHB27I under cofactor/analogue-induced ‘star’ activity conditions. E. coli and λ genomic DNAs were digested with TthHB27I under cofactor/analogue-induced ‘star’ conditions, while varying: temperature, incubation time and TthHB27I amount. 0.5 μg of DNAs were digested with TthHB27I in REase buffer supplemented with 100 μM SIN and 25% (v/v) DMSO. a Cleavage control by reaction temperature. Reactions conducted with E. coli DNA digested with 2 U of TthHB27I for 1 h at temperatures from 30 °C to 80 °C. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction at 30 °C; lane 2, 35 °C; lane 3, 40 °C; lane 4, 45 °C; lane 5, 50 °C; lane 6, 55 °C; lane 7, 60 °C; lane 8, 65 °C; lane 9, 70 °C; lane 10, 75 °C; lane 11, 80 °C. b The same as ( a ), except that λ DNA was used. c Cleavage control by reaction time. E. coli DNA was digested at 65 °C at times ranging from 7.5 min to 16 h. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction conducted for 7.5 min; lane 2, 15 min; lane 3, 30 min; lane 4, 1 h; lane 5, 2 h; lane 6, 4 h; lane 7, 8 h; lane 8, 16 h. d The same as ( c ), except that λ DNA was used. e Cleavage control by the enzyme amount. E. coli DNA was digested at 65 °C with TthHB27I. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, DNA digested with 8 U TthHB27I; lane 2, 4 U; lane 3, 2 U; lane 4, 1 U; lane 5, 0.5 U; lane 6, 0.25 U; lane 7, 0.12 U; lane 8, 0.06 U. f The same as ( e ), except that λ DNA was used

    Journal: BMC Genomics

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

    doi: 10.1186/s12864-018-4748-0

    Figure Lengend Snippet: Controlling of partial cleavage of genomic DNAs by TthHB27I under cofactor/analogue-induced ‘star’ activity conditions. E. coli and λ genomic DNAs were digested with TthHB27I under cofactor/analogue-induced ‘star’ conditions, while varying: temperature, incubation time and TthHB27I amount. 0.5 μg of DNAs were digested with TthHB27I in REase buffer supplemented with 100 μM SIN and 25% (v/v) DMSO. a Cleavage control by reaction temperature. Reactions conducted with E. coli DNA digested with 2 U of TthHB27I for 1 h at temperatures from 30 °C to 80 °C. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction at 30 °C; lane 2, 35 °C; lane 3, 40 °C; lane 4, 45 °C; lane 5, 50 °C; lane 6, 55 °C; lane 7, 60 °C; lane 8, 65 °C; lane 9, 70 °C; lane 10, 75 °C; lane 11, 80 °C. b The same as ( a ), except that λ DNA was used. c Cleavage control by reaction time. E. coli DNA was digested at 65 °C at times ranging from 7.5 min to 16 h. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction conducted for 7.5 min; lane 2, 15 min; lane 3, 30 min; lane 4, 1 h; lane 5, 2 h; lane 6, 4 h; lane 7, 8 h; lane 8, 16 h. d The same as ( c ), except that λ DNA was used. e Cleavage control by the enzyme amount. E. coli DNA was digested at 65 °C with TthHB27I. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, DNA digested with 8 U TthHB27I; lane 2, 4 U; lane 3, 2 U; lane 4, 1 U; lane 5, 0.5 U; lane 6, 0.25 U; lane 7, 0.12 U; lane 8, 0.06 U. f The same as ( e ), except that λ DNA was used

    Article Snippet: DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania).

    Techniques: Activity Assay, Incubation

    MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression ( A ) or downregulation ( B ) was then checked by qRT-PCR and compared to that of their respective controls in MCF7 cells. ( C , D ) MED1 was overexpressed ( C ) or inhibited ( D ) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small RNA. The graphical data points represent mean + S.D of at least three independent experiments (**P

    Journal: Scientific Reports

    Article Title: Essential role of MED1 in the transcriptional regulation of ER-dependent oncogenic miRNAs in breast cancer

    doi: 10.1038/s41598-018-29546-9

    Figure Lengend Snippet: MED1 is involved in the regulation of miRNAs in breast cancer. Confirmation of overexpression/inhibition of MED1 transcript in MCF7 cells. MED1 was transiently up/downregulated by transfecting MCF7 cells with pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) and esiMED1/esiCtrl. The corresponding effect on MED1 transcript levels in response to MED1 overexpression ( A ) or downregulation ( B ) was then checked by qRT-PCR and compared to that of their respective controls in MCF7 cells. ( C , D ) MED1 was overexpressed ( C ) or inhibited ( D ) in MCF7 cells and stem loop qRT-PCR was done to check the levels of various breast cancer related miRNAs. The mRNA data was normalized to GAPDH and miRNA data was normalized to U6 small RNA. The graphical data points represent mean + S.D of at least three independent experiments (**P

    Article Snippet: RNA isolation and Stem-loop qRT-PCR Total RNA isolation from cell lines was done using RNA Isolation kit (GeneJET RNA purification kit, Thermo Scientific), according to manufacturer’s instructions.

    Techniques: Over Expression, Inhibition, Quantitative RT-PCR

    Effect of ER-α and MED1 on transcriptional regulation of miR-191/425 cluster. ( A–C ) MED1 or ER-α were overexpressed or inhibited and stem loop qRT-PCR was done to check the levels of miR-191 and miR-425 ( A ). Estrogen (E2) or vehicle (ethanol, Eth) treatment was given along with esiMED1 or tamoxifen (Tm) and the effect on miR-191/425 was observed using stem loop qRT-PCR. ( B , C ) The data was normalized to U6 small RNA (RNU6B) using 2 −ΔCt method. ( D ) Levels of MED1 were transiently modulated using pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) in a panel of ER+ve/-ve breast cancer cell lines (MCF7, ZR75-1, MDA-MB-231) and effect on miR-191/425 levels was tested using stem-loop qRT PCR. Induced levels of both the cluster miRNAs (miR-191 and miR-425) were observed in response to MED1 overexpression in ER+ve cell lines (MCF7, ZR75-1) while opposite results were observed for ER-ve cell line (MDA-MB-231). Thereby confirming that MED1 mediated induction of miR-191/425 occurs in an ER-dependent manner. The graphical data points represent mean + S.D of at least three independent experiments (**P

    Journal: Scientific Reports

    Article Title: Essential role of MED1 in the transcriptional regulation of ER-dependent oncogenic miRNAs in breast cancer

    doi: 10.1038/s41598-018-29546-9

    Figure Lengend Snippet: Effect of ER-α and MED1 on transcriptional regulation of miR-191/425 cluster. ( A–C ) MED1 or ER-α were overexpressed or inhibited and stem loop qRT-PCR was done to check the levels of miR-191 and miR-425 ( A ). Estrogen (E2) or vehicle (ethanol, Eth) treatment was given along with esiMED1 or tamoxifen (Tm) and the effect on miR-191/425 was observed using stem loop qRT-PCR. ( B , C ) The data was normalized to U6 small RNA (RNU6B) using 2 −ΔCt method. ( D ) Levels of MED1 were transiently modulated using pCDNA3.1-MED1 (MED1) or pCDNA3.1 (PC) in a panel of ER+ve/-ve breast cancer cell lines (MCF7, ZR75-1, MDA-MB-231) and effect on miR-191/425 levels was tested using stem-loop qRT PCR. Induced levels of both the cluster miRNAs (miR-191 and miR-425) were observed in response to MED1 overexpression in ER+ve cell lines (MCF7, ZR75-1) while opposite results were observed for ER-ve cell line (MDA-MB-231). Thereby confirming that MED1 mediated induction of miR-191/425 occurs in an ER-dependent manner. The graphical data points represent mean + S.D of at least three independent experiments (**P

    Article Snippet: RNA isolation and Stem-loop qRT-PCR Total RNA isolation from cell lines was done using RNA Isolation kit (GeneJET RNA purification kit, Thermo Scientific), according to manufacturer’s instructions.

    Techniques: Quantitative RT-PCR, Multiple Displacement Amplification, Over Expression

    Efficiency of DNA removal by DNaseI and its fusion variants during RNA purification procedure. DNaseDT denotes the fusion with the (HhH) 2 domain of DNase from an extremely salt tolerant bacterium Thioalkalivibrio sp. K90mix , DNaseBS denotes the fusion with homologous domain of ComEA protein from Bacillus subtilis . The upper picture represents quantitative evaluation of undigested DNA remaining in eluates (RT-qPCR without added reverse transcriptase). The lower picture represents RT-qPCR results obtained using the same eluates. In the both pictures labels on the horizontal axis indicate the used nuclease, amount of it and the dilution ratio of the eluates before reverse transcription step and quantitation of DNA. A modified protocol of GeneJET™ Whole Blood RNA Purification Mini Kit (Thermo Fisher Scientific, #K0761) was followed and RNA purification columns supplied by the manufacturer were used. During the experiment we have purified total blood RNA. Three arbitrary blood samples were analysed. DNA digestion was performed directly on a filter of a RNA purification column, were respective DNase enzyme was loaded.

    Journal: PLoS ONE

    Article Title: Enhancement of DNaseI Salt Tolerance by Mimicking the Domain Structure of DNase from an Extremely Halotolerant Bacterium Thioalkalivibrio sp. K90mix

    doi: 10.1371/journal.pone.0150404

    Figure Lengend Snippet: Efficiency of DNA removal by DNaseI and its fusion variants during RNA purification procedure. DNaseDT denotes the fusion with the (HhH) 2 domain of DNase from an extremely salt tolerant bacterium Thioalkalivibrio sp. K90mix , DNaseBS denotes the fusion with homologous domain of ComEA protein from Bacillus subtilis . The upper picture represents quantitative evaluation of undigested DNA remaining in eluates (RT-qPCR without added reverse transcriptase). The lower picture represents RT-qPCR results obtained using the same eluates. In the both pictures labels on the horizontal axis indicate the used nuclease, amount of it and the dilution ratio of the eluates before reverse transcription step and quantitation of DNA. A modified protocol of GeneJET™ Whole Blood RNA Purification Mini Kit (Thermo Fisher Scientific, #K0761) was followed and RNA purification columns supplied by the manufacturer were used. During the experiment we have purified total blood RNA. Three arbitrary blood samples were analysed. DNA digestion was performed directly on a filter of a RNA purification column, were respective DNase enzyme was loaded.

    Article Snippet: Elimination of contaminant DNA during RNA purification RNA was purified from three arbitrary human blood samples using GeneJET™ Whole Blood RNA Purification Mini Kit (Thermo Fisher Scientific, #K0761).

    Techniques: Purification, Quantitative RT-PCR, Quantitation Assay, Modification