genejet pcr purification kit  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier
Bioz Manufacturer Symbol Thermo Fisher manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Name:
    GeneJET PCR Purification Kit
    Description:
    Thermo Scientific GeneJET PCR Purification Kit utilizes a proprietary silica based membrane technology in the form of a convenient spin column eliminating the need for tedious resin manipulations or toxic phenol chloroform extractions It effectively removes primers dNTPs unincorporated labeled nucleotides enzymes and salts from PCR and other reaction mixtures The kit can be used for purification of DNA fragments from 25 bp to 20 kb with recovery rates up to 100 Each GeneJET purification column has a total binding capacity of up to 25 µg of DNA and the entire procedure takes 5 minutes The purified DNA can be used in common downstream applications such as sequencing restriction digestion labeling ligation cloning in vitro transcription blotting or in situ hybridization Highlights• Highly efficient 90 to 100 recoveries in the range of 100 bp to 10 kb• Fast procedure takes only 5 minutes• Convenient spin columns are capped and assembled with collection tubesApplications• Fast and efficient purification of DNA fragments ideal for use in all conventional molecular biology procedures including • FastDigest or conventional restriction digestion• Automated fluorescent or radioactive sequencing• PCR• In vitro transcription• In situ hybridization• Labeling• Cloning
    Catalog Number:
    K0701
    Price:
    None
    Category:
    Kits and Assays
    Applications:
    DNA & RNA Purification & Analysis|DNA Extraction|PCR Product Clean-Up
    Buy from Supplier


    Structured Review

    Thermo Fisher genejet pcr purification kit
    Thermo Scientific GeneJET PCR Purification Kit utilizes a proprietary silica based membrane technology in the form of a convenient spin column eliminating the need for tedious resin manipulations or toxic phenol chloroform extractions It effectively removes primers dNTPs unincorporated labeled nucleotides enzymes and salts from PCR and other reaction mixtures The kit can be used for purification of DNA fragments from 25 bp to 20 kb with recovery rates up to 100 Each GeneJET purification column has a total binding capacity of up to 25 µg of DNA and the entire procedure takes 5 minutes The purified DNA can be used in common downstream applications such as sequencing restriction digestion labeling ligation cloning in vitro transcription blotting or in situ hybridization Highlights• Highly efficient 90 to 100 recoveries in the range of 100 bp to 10 kb• Fast procedure takes only 5 minutes• Convenient spin columns are capped and assembled with collection tubesApplications• Fast and efficient purification of DNA fragments ideal for use in all conventional molecular biology procedures including • FastDigest or conventional restriction digestion• Automated fluorescent or radioactive sequencing• PCR• In vitro transcription• In situ hybridization• Labeling• Cloning
    https://www.bioz.com/result/genejet pcr purification kit/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    genejet pcr purification kit - by Bioz Stars, 2021-05
    97/100 stars

    Images

    Related Articles

    Purification:

    Article Title: Arabidopsis RRP6L1 and RRP6L2 Function in FLOWERING LOCUS C Silencing via Regulation of Antisense RNA Synthesis
    Article Snippet: .. The ChIPed DNA was purified using PCR purification kit (Fermentas) and qPCR was performed. ..

    Article Title: TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens
    Article Snippet: 10 μl of the RT-mix was added to each tube of RNA + primer, which was incubated 10 min at room temperature, then 50 min at 42 °C, and finally 30 min at 65 °C (to inactivate enzymes). .. Finally, the cDNA was purified using a PCR-purification kit (GeneJET Gel Extraction Kit, Thermo Scientific, Milian, Vernier, Switzerland), and eluted in 50 μl Elution Buffer. .. Second-strand PCR To generate double stranded DNA, with the appropriate adaptors for Illumina sequencing, as well as addition of a four nucleotide EMOTE barcode which serves to identify the RNA sample and pool, a PCR reaction was prepared for each RT-reaction: 10 μl PCR-purified RT-reaction, 27 μl water, 10 μl Q5 Polymerase buffer, 1.5 μl dNTP (2.5 mM each), 1.5 μl 100nM of one of primers D6A, D6B, D6C, etc. (each with a unique EMOTE barcode, Table ), 1.5 μl 10 uM Primer B-PE-PCR20, and 0.5 μl Q5® Hot Start High-Fidelity DNA Polymerase (NEB).

    Article Title: HIV-1 Interacts with Human Endogenous Retrovirus K (HML-2) Envelopes Derived from Human Primary Lymphocytes
    Article Snippet: The Env-specific RT primer sequence represents the majority consensus of the HERV-Ks with nucleotide sequence within the selected region. .. To eliminate the interference of the reverse transcription primer on the subsequent PCR amplification reaction, HERV-K env RT products were purified using a PCR purification kit (Invitrogen) per the manufacturer's instructions. .. One-tenth of the purified reverse transcription reaction was used to amplify the 700 bp of the HERV-K env SU region used for expression profiling.

    Article Title: Replication-Transcription Conflicts Generate R-Loops that Orchestrate Bacterial Stress Survival and Pathogenesis
    Article Snippet: .. pHM64: The rnhC gene was amplified using primers HM464/HM517 and purified using a PCR purification kit (Thermo). .. The plasmid pCAL838 was linearized with NheI and HindIII and gel purified using a PCR purification kit (Thermo).

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application
    Article Snippet: .. The Wizard® SV Gel and PCR Clean-Up System (Promega; Pr), the GeneJET PCR Purification Kit (Thermo Fisher Scientific; Th), the PureLink® PCR Purification Kit (Invitrogen; In) and the Chromatin IP DNA Purification Kit (Active Motif; Am) performed poorly with de-crosslinked chromatin. .. Consistently, these reagents recovered less than 50% of input DNA with purified DNA even when expected DNA amounts were relatively high (10–50 ng).

    Article Title: Cholecystokinin attenuates radiation-induced lung cancer cell apoptosis by modulating p53 gene transcription
    Article Snippet: After centrifugation (3000 rpm, 10 min), the supernatant was incubated with antibodies of interest (or isotype IgG) overnight at 4°C, followed by addition of protein G agarose beads and incubated for another 2 h. The beads were collected by centrifugation (3000 rpm, 10 min); the attached immune complexes were eluted with an eluting buffer. .. The DNA was recovered by a PCR purification kit (Invitrogen, Carlsbad, CA) and analyzed by qPCR. ..

    Article Title: Gene cloning, heterologous expression, and partial characterization of a novel cold-adapted subfamily I.3 lipase from Pseudomonas fluorescence KE38
    Article Snippet: General methods Transformation of E. coli with plasmid DNA, agarose gel electrophoresis, restriction endonuclease digestions, DNA ligation with T4 DNA ligase, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Sambrook et al. . .. Commercial kits were used for the isolation of plasmid and genomic DNAs (GeneJET plasmid mini prep and Genomic DNA purification kits, Thermo Scientific), and purification of DNA fragments (GeneJET gel extraction, and PCR purification kits, Thermo Scientific) according to the manufacturers’ instructions. .. For DNA sequencing, facilities at the Biotechnology and Bioengineering Application and Research Center (BIYOMER) in Izmir Institute of Technology were used.

    Article Title: The Role of the Arabidopsis Exosome in siRNA-Independent Silencing of Heterochromatic Loci
    Article Snippet: .. The ChIPed DNA was purified using PCR purification kit (Fermentas) before being used for qPCR. ..

    Polymerase Chain Reaction:

    Article Title: Arabidopsis RRP6L1 and RRP6L2 Function in FLOWERING LOCUS C Silencing via Regulation of Antisense RNA Synthesis
    Article Snippet: .. The ChIPed DNA was purified using PCR purification kit (Fermentas) and qPCR was performed. ..

    Article Title: TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens
    Article Snippet: 10 μl of the RT-mix was added to each tube of RNA + primer, which was incubated 10 min at room temperature, then 50 min at 42 °C, and finally 30 min at 65 °C (to inactivate enzymes). .. Finally, the cDNA was purified using a PCR-purification kit (GeneJET Gel Extraction Kit, Thermo Scientific, Milian, Vernier, Switzerland), and eluted in 50 μl Elution Buffer. .. Second-strand PCR To generate double stranded DNA, with the appropriate adaptors for Illumina sequencing, as well as addition of a four nucleotide EMOTE barcode which serves to identify the RNA sample and pool, a PCR reaction was prepared for each RT-reaction: 10 μl PCR-purified RT-reaction, 27 μl water, 10 μl Q5 Polymerase buffer, 1.5 μl dNTP (2.5 mM each), 1.5 μl 100nM of one of primers D6A, D6B, D6C, etc. (each with a unique EMOTE barcode, Table ), 1.5 μl 10 uM Primer B-PE-PCR20, and 0.5 μl Q5® Hot Start High-Fidelity DNA Polymerase (NEB).

    Article Title: HIV-1 Interacts with Human Endogenous Retrovirus K (HML-2) Envelopes Derived from Human Primary Lymphocytes
    Article Snippet: The Env-specific RT primer sequence represents the majority consensus of the HERV-Ks with nucleotide sequence within the selected region. .. To eliminate the interference of the reverse transcription primer on the subsequent PCR amplification reaction, HERV-K env RT products were purified using a PCR purification kit (Invitrogen) per the manufacturer's instructions. .. One-tenth of the purified reverse transcription reaction was used to amplify the 700 bp of the HERV-K env SU region used for expression profiling.

    Article Title: Replication-Transcription Conflicts Generate R-Loops that Orchestrate Bacterial Stress Survival and Pathogenesis
    Article Snippet: .. pHM64: The rnhC gene was amplified using primers HM464/HM517 and purified using a PCR purification kit (Thermo). .. The plasmid pCAL838 was linearized with NheI and HindIII and gel purified using a PCR purification kit (Thermo).

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application
    Article Snippet: .. The Wizard® SV Gel and PCR Clean-Up System (Promega; Pr), the GeneJET PCR Purification Kit (Thermo Fisher Scientific; Th), the PureLink® PCR Purification Kit (Invitrogen; In) and the Chromatin IP DNA Purification Kit (Active Motif; Am) performed poorly with de-crosslinked chromatin. .. Consistently, these reagents recovered less than 50% of input DNA with purified DNA even when expected DNA amounts were relatively high (10–50 ng).

    Article Title: Cholecystokinin attenuates radiation-induced lung cancer cell apoptosis by modulating p53 gene transcription
    Article Snippet: After centrifugation (3000 rpm, 10 min), the supernatant was incubated with antibodies of interest (or isotype IgG) overnight at 4°C, followed by addition of protein G agarose beads and incubated for another 2 h. The beads were collected by centrifugation (3000 rpm, 10 min); the attached immune complexes were eluted with an eluting buffer. .. The DNA was recovered by a PCR purification kit (Invitrogen, Carlsbad, CA) and analyzed by qPCR. ..

    Article Title: Gene cloning, heterologous expression, and partial characterization of a novel cold-adapted subfamily I.3 lipase from Pseudomonas fluorescence KE38
    Article Snippet: General methods Transformation of E. coli with plasmid DNA, agarose gel electrophoresis, restriction endonuclease digestions, DNA ligation with T4 DNA ligase, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Sambrook et al. . .. Commercial kits were used for the isolation of plasmid and genomic DNAs (GeneJET plasmid mini prep and Genomic DNA purification kits, Thermo Scientific), and purification of DNA fragments (GeneJET gel extraction, and PCR purification kits, Thermo Scientific) according to the manufacturers’ instructions. .. For DNA sequencing, facilities at the Biotechnology and Bioengineering Application and Research Center (BIYOMER) in Izmir Institute of Technology were used.

    Article Title: The Role of the Arabidopsis Exosome in siRNA-Independent Silencing of Heterochromatic Loci
    Article Snippet: .. The ChIPed DNA was purified using PCR purification kit (Fermentas) before being used for qPCR. ..

    Real-time Polymerase Chain Reaction:

    Article Title: Arabidopsis RRP6L1 and RRP6L2 Function in FLOWERING LOCUS C Silencing via Regulation of Antisense RNA Synthesis
    Article Snippet: .. The ChIPed DNA was purified using PCR purification kit (Fermentas) and qPCR was performed. ..

    Article Title: Cholecystokinin attenuates radiation-induced lung cancer cell apoptosis by modulating p53 gene transcription
    Article Snippet: After centrifugation (3000 rpm, 10 min), the supernatant was incubated with antibodies of interest (or isotype IgG) overnight at 4°C, followed by addition of protein G agarose beads and incubated for another 2 h. The beads were collected by centrifugation (3000 rpm, 10 min); the attached immune complexes were eluted with an eluting buffer. .. The DNA was recovered by a PCR purification kit (Invitrogen, Carlsbad, CA) and analyzed by qPCR. ..

    Article Title: The Role of the Arabidopsis Exosome in siRNA-Independent Silencing of Heterochromatic Loci
    Article Snippet: .. The ChIPed DNA was purified using PCR purification kit (Fermentas) before being used for qPCR. ..

    Gel Extraction:

    Article Title: TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens
    Article Snippet: 10 μl of the RT-mix was added to each tube of RNA + primer, which was incubated 10 min at room temperature, then 50 min at 42 °C, and finally 30 min at 65 °C (to inactivate enzymes). .. Finally, the cDNA was purified using a PCR-purification kit (GeneJET Gel Extraction Kit, Thermo Scientific, Milian, Vernier, Switzerland), and eluted in 50 μl Elution Buffer. .. Second-strand PCR To generate double stranded DNA, with the appropriate adaptors for Illumina sequencing, as well as addition of a four nucleotide EMOTE barcode which serves to identify the RNA sample and pool, a PCR reaction was prepared for each RT-reaction: 10 μl PCR-purified RT-reaction, 27 μl water, 10 μl Q5 Polymerase buffer, 1.5 μl dNTP (2.5 mM each), 1.5 μl 100nM of one of primers D6A, D6B, D6C, etc. (each with a unique EMOTE barcode, Table ), 1.5 μl 10 uM Primer B-PE-PCR20, and 0.5 μl Q5® Hot Start High-Fidelity DNA Polymerase (NEB).

    Article Title: Gene cloning, heterologous expression, and partial characterization of a novel cold-adapted subfamily I.3 lipase from Pseudomonas fluorescence KE38
    Article Snippet: General methods Transformation of E. coli with plasmid DNA, agarose gel electrophoresis, restriction endonuclease digestions, DNA ligation with T4 DNA ligase, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Sambrook et al. . .. Commercial kits were used for the isolation of plasmid and genomic DNAs (GeneJET plasmid mini prep and Genomic DNA purification kits, Thermo Scientific), and purification of DNA fragments (GeneJET gel extraction, and PCR purification kits, Thermo Scientific) according to the manufacturers’ instructions. .. For DNA sequencing, facilities at the Biotechnology and Bioengineering Application and Research Center (BIYOMER) in Izmir Institute of Technology were used.

    Amplification:

    Article Title: HIV-1 Interacts with Human Endogenous Retrovirus K (HML-2) Envelopes Derived from Human Primary Lymphocytes
    Article Snippet: The Env-specific RT primer sequence represents the majority consensus of the HERV-Ks with nucleotide sequence within the selected region. .. To eliminate the interference of the reverse transcription primer on the subsequent PCR amplification reaction, HERV-K env RT products were purified using a PCR purification kit (Invitrogen) per the manufacturer's instructions. .. One-tenth of the purified reverse transcription reaction was used to amplify the 700 bp of the HERV-K env SU region used for expression profiling.

    Article Title: Replication-Transcription Conflicts Generate R-Loops that Orchestrate Bacterial Stress Survival and Pathogenesis
    Article Snippet: .. pHM64: The rnhC gene was amplified using primers HM464/HM517 and purified using a PCR purification kit (Thermo). .. The plasmid pCAL838 was linearized with NheI and HindIII and gel purified using a PCR purification kit (Thermo).

    Chromatin Immunoprecipitation:

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application
    Article Snippet: .. The Wizard® SV Gel and PCR Clean-Up System (Promega; Pr), the GeneJET PCR Purification Kit (Thermo Fisher Scientific; Th), the PureLink® PCR Purification Kit (Invitrogen; In) and the Chromatin IP DNA Purification Kit (Active Motif; Am) performed poorly with de-crosslinked chromatin. .. Consistently, these reagents recovered less than 50% of input DNA with purified DNA even when expected DNA amounts were relatively high (10–50 ng).

    DNA Purification:

    Article Title: Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application
    Article Snippet: .. The Wizard® SV Gel and PCR Clean-Up System (Promega; Pr), the GeneJET PCR Purification Kit (Thermo Fisher Scientific; Th), the PureLink® PCR Purification Kit (Invitrogen; In) and the Chromatin IP DNA Purification Kit (Active Motif; Am) performed poorly with de-crosslinked chromatin. .. Consistently, these reagents recovered less than 50% of input DNA with purified DNA even when expected DNA amounts were relatively high (10–50 ng).

    Article Title: Gene cloning, heterologous expression, and partial characterization of a novel cold-adapted subfamily I.3 lipase from Pseudomonas fluorescence KE38
    Article Snippet: General methods Transformation of E. coli with plasmid DNA, agarose gel electrophoresis, restriction endonuclease digestions, DNA ligation with T4 DNA ligase, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Sambrook et al. . .. Commercial kits were used for the isolation of plasmid and genomic DNAs (GeneJET plasmid mini prep and Genomic DNA purification kits, Thermo Scientific), and purification of DNA fragments (GeneJET gel extraction, and PCR purification kits, Thermo Scientific) according to the manufacturers’ instructions. .. For DNA sequencing, facilities at the Biotechnology and Bioengineering Application and Research Center (BIYOMER) in Izmir Institute of Technology were used.

    Isolation:

    Article Title: Gene cloning, heterologous expression, and partial characterization of a novel cold-adapted subfamily I.3 lipase from Pseudomonas fluorescence KE38
    Article Snippet: General methods Transformation of E. coli with plasmid DNA, agarose gel electrophoresis, restriction endonuclease digestions, DNA ligation with T4 DNA ligase, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Sambrook et al. . .. Commercial kits were used for the isolation of plasmid and genomic DNAs (GeneJET plasmid mini prep and Genomic DNA purification kits, Thermo Scientific), and purification of DNA fragments (GeneJET gel extraction, and PCR purification kits, Thermo Scientific) according to the manufacturers’ instructions. .. For DNA sequencing, facilities at the Biotechnology and Bioengineering Application and Research Center (BIYOMER) in Izmir Institute of Technology were used.

    Plasmid Preparation:

    Article Title: Gene cloning, heterologous expression, and partial characterization of a novel cold-adapted subfamily I.3 lipase from Pseudomonas fluorescence KE38
    Article Snippet: General methods Transformation of E. coli with plasmid DNA, agarose gel electrophoresis, restriction endonuclease digestions, DNA ligation with T4 DNA ligase, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Sambrook et al. . .. Commercial kits were used for the isolation of plasmid and genomic DNAs (GeneJET plasmid mini prep and Genomic DNA purification kits, Thermo Scientific), and purification of DNA fragments (GeneJET gel extraction, and PCR purification kits, Thermo Scientific) according to the manufacturers’ instructions. .. For DNA sequencing, facilities at the Biotechnology and Bioengineering Application and Research Center (BIYOMER) in Izmir Institute of Technology were used.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Thermo Fisher rna isolation kit
    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
    Rna Isolation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna isolation kit/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rna isolation kit - by Bioz Stars, 2021-05
    97/100 stars
      Buy from Supplier

    97
    Thermo Fisher pcr purification kit
    Real time quantitative <t>RT-PCR</t> <t>(RT-qPCR)</t>
    Pcr Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr purification kit/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcr purification kit - by Bioz Stars, 2021-05
    97/100 stars
      Buy from Supplier

    Image Search Results


    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

    Real time quantitative RT-PCR (RT-qPCR)

    Journal: American Journal of Translational Research

    Article Title: Cholecystokinin attenuates radiation-induced lung cancer cell apoptosis by modulating p53 gene transcription

    doi:

    Figure Lengend Snippet: Real time quantitative RT-PCR (RT-qPCR)

    Article Snippet: The DNA was recovered by a PCR purification kit (Invitrogen, Carlsbad, CA) and analyzed by qPCR.

    Techniques: Quantitative RT-PCR

    The rrp6l1-3 and rrp6l2-3 mutants affect flowering time and gene expression. (A) The late-flowering phenotype of rrp6l1 and rrp6l2 mutants grown under long day conditions. Flowering time was measured as rosette leaf number at bolting. To control for effects of ecotype, the phenotype of mutants was compared to wild-type plants of Col-0 and Ws ecotypes. Error bars represent standard deviation (SD). (B) The late-flowering phenotype of rrp6l1-3 rrp6l2-3 mutants grown under short day conditions. 66-day-old plants are shown. (C) Effect of rrp6l1-3 and rrp6l2-3 mutations on the expression of the FLC , SOC1 and FT . RT-PCR showed that the rrp6l1-3 rrp6l2-3 double mutant ( rrp6l1/2 ), has increased expression of FLC and decreased expression of SOC1 and FT , which act downstream of FLC . (-RT) is the no reverse transcriptase control. (D) The expression of FCA , FPA , FLD , FVE , FLM , and LDL2 , genes involved in regulation of flowering time in the autonomous flowering pathway, is not affected in rrp6l1/2 mutants. (E) FLC expression is not affected in AtCSL4 -2 T-DNA mutant and RRP41 iRNAi line. RRP41 corresponds to the iRNAi line grown without estradiol, and rrp41-i corresponds to line grown on estradiol-containing medium, to induce the RNAi-mediated knockdown of RRP41 . TUBULIN 2 and ACTIN 7 were used as loading controls.

    Journal: PLoS Genetics

    Article Title: Arabidopsis RRP6L1 and RRP6L2 Function in FLOWERING LOCUS C Silencing via Regulation of Antisense RNA Synthesis

    doi: 10.1371/journal.pgen.1004612

    Figure Lengend Snippet: The rrp6l1-3 and rrp6l2-3 mutants affect flowering time and gene expression. (A) The late-flowering phenotype of rrp6l1 and rrp6l2 mutants grown under long day conditions. Flowering time was measured as rosette leaf number at bolting. To control for effects of ecotype, the phenotype of mutants was compared to wild-type plants of Col-0 and Ws ecotypes. Error bars represent standard deviation (SD). (B) The late-flowering phenotype of rrp6l1-3 rrp6l2-3 mutants grown under short day conditions. 66-day-old plants are shown. (C) Effect of rrp6l1-3 and rrp6l2-3 mutations on the expression of the FLC , SOC1 and FT . RT-PCR showed that the rrp6l1-3 rrp6l2-3 double mutant ( rrp6l1/2 ), has increased expression of FLC and decreased expression of SOC1 and FT , which act downstream of FLC . (-RT) is the no reverse transcriptase control. (D) The expression of FCA , FPA , FLD , FVE , FLM , and LDL2 , genes involved in regulation of flowering time in the autonomous flowering pathway, is not affected in rrp6l1/2 mutants. (E) FLC expression is not affected in AtCSL4 -2 T-DNA mutant and RRP41 iRNAi line. RRP41 corresponds to the iRNAi line grown without estradiol, and rrp41-i corresponds to line grown on estradiol-containing medium, to induce the RNAi-mediated knockdown of RRP41 . TUBULIN 2 and ACTIN 7 were used as loading controls.

    Article Snippet: The ChIPed DNA was purified using PCR purification kit (Fermentas) and qPCR was performed.

    Techniques: Expressing, Standard Deviation, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Activated Clotting Time Assay

    TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule

    Journal: BMC Genomics

    Article Title: TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens

    doi: 10.1186/s12864-016-3211-3

    Figure Lengend Snippet: TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule

    Article Snippet: Finally, the cDNA was purified using a PCR-purification kit (GeneJET Gel Extraction Kit, Thermo Scientific, Milian, Vernier, Switzerland), and eluted in 50 μl Elution Buffer.

    Techniques: In Silico, Purification, Ligation, Sequencing, Polymerase Chain Reaction, RNA Sequencing Assay, Software