taqman fast universal pcr master mix  (Thermo Fisher)


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    Thermo Fisher taqman fast universal pcr master mix
    Detection of pp71 RNA and protein in primary glioma specimens. A: RNA was extracted from 20 different primary brain tissues, synthesized into cDNA, and amplified using pp71 and Rab14-specific <t>PCR</t> primers. RNA from U251 glioma cells mock infected or infected with HCMV Towne strain and commercially available RNA samples from normal fetal and adult human brain were used as controls. The N.C. negative control PCR contained water in place of cDNA. B : Several cDNA samples described in A were analyzed by <t>TaqMan</t> using primers and probes specific for the pp71 gene and normalized to Rab14. Copy number was determined using Ad169 viral DNA standard curve. C : Western blot analysis for pp71 from 10 different primary brain tissues. Lysates from normal human astrocytes and mock-infected or HCMV-infected U87 glioma cells were used as controls. D : Cells from 3 freshly resected GBM were sorted using CD133 labeled antibody and analyzed by RT-PCR for pp71 and Rab14. Negative control (NC) is PCR performed with water instead of cDNA. E : HCMV-infected primary GBM cell line (3832) was sorted using CD133 labeled antibody at 72 hours post-infection and analyzed by TaqMan for pp71 compared to a mock-treated control. The relative expression of pp71 normalized to Rab14 is displayed for 3 independent experiments, *p
    Taqman Fast Universal Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 313 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Cytomegalovirus pp71 Protein Is Expressed in Human Glioblastoma and Promotes Pro-Angiogenic Signaling by Activation of Stem Cell Factor"

    Article Title: Cytomegalovirus pp71 Protein Is Expressed in Human Glioblastoma and Promotes Pro-Angiogenic Signaling by Activation of Stem Cell Factor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068176

    Detection of pp71 RNA and protein in primary glioma specimens. A: RNA was extracted from 20 different primary brain tissues, synthesized into cDNA, and amplified using pp71 and Rab14-specific PCR primers. RNA from U251 glioma cells mock infected or infected with HCMV Towne strain and commercially available RNA samples from normal fetal and adult human brain were used as controls. The N.C. negative control PCR contained water in place of cDNA. B : Several cDNA samples described in A were analyzed by TaqMan using primers and probes specific for the pp71 gene and normalized to Rab14. Copy number was determined using Ad169 viral DNA standard curve. C : Western blot analysis for pp71 from 10 different primary brain tissues. Lysates from normal human astrocytes and mock-infected or HCMV-infected U87 glioma cells were used as controls. D : Cells from 3 freshly resected GBM were sorted using CD133 labeled antibody and analyzed by RT-PCR for pp71 and Rab14. Negative control (NC) is PCR performed with water instead of cDNA. E : HCMV-infected primary GBM cell line (3832) was sorted using CD133 labeled antibody at 72 hours post-infection and analyzed by TaqMan for pp71 compared to a mock-treated control. The relative expression of pp71 normalized to Rab14 is displayed for 3 independent experiments, *p
    Figure Legend Snippet: Detection of pp71 RNA and protein in primary glioma specimens. A: RNA was extracted from 20 different primary brain tissues, synthesized into cDNA, and amplified using pp71 and Rab14-specific PCR primers. RNA from U251 glioma cells mock infected or infected with HCMV Towne strain and commercially available RNA samples from normal fetal and adult human brain were used as controls. The N.C. negative control PCR contained water in place of cDNA. B : Several cDNA samples described in A were analyzed by TaqMan using primers and probes specific for the pp71 gene and normalized to Rab14. Copy number was determined using Ad169 viral DNA standard curve. C : Western blot analysis for pp71 from 10 different primary brain tissues. Lysates from normal human astrocytes and mock-infected or HCMV-infected U87 glioma cells were used as controls. D : Cells from 3 freshly resected GBM were sorted using CD133 labeled antibody and analyzed by RT-PCR for pp71 and Rab14. Negative control (NC) is PCR performed with water instead of cDNA. E : HCMV-infected primary GBM cell line (3832) was sorted using CD133 labeled antibody at 72 hours post-infection and analyzed by TaqMan for pp71 compared to a mock-treated control. The relative expression of pp71 normalized to Rab14 is displayed for 3 independent experiments, *p

    Techniques Used: Synthesized, Amplification, Polymerase Chain Reaction, Infection, Negative Control, Western Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Expressing

    2) Product Images from "Characterization of the canine mda-7 gene, transcripts and expression patterns"

    Article Title: Characterization of the canine mda-7 gene, transcripts and expression patterns

    Journal:

    doi: 10.1016/j.gene.2014.05.054

    Quantitative RT-PCR probe locations. TaqMan probes were designed against transcript specific sequences to detect sv2, sv3, sv4 and sv5. The copy number of canine mda- 7sv1 was deduced by subtracting the sum of the copy number of sv2, sv3, sv4 and sv5 from
    Figure Legend Snippet: Quantitative RT-PCR probe locations. TaqMan probes were designed against transcript specific sequences to detect sv2, sv3, sv4 and sv5. The copy number of canine mda- 7sv1 was deduced by subtracting the sum of the copy number of sv2, sv3, sv4 and sv5 from

    Techniques Used: Quantitative RT-PCR, Multiple Displacement Amplification

    3) Product Images from "Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA"

    Article Title: Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.05.014

    Editing of CEP290 Transcripts Occurs in HEK293T after Transfection with PTMs (A) Illustration of adeno-associated virus genome arrangement of a CEP290 5′ PTM with binding domain 07 (PTM_07) with either no poly-adenylation signal (NPA) or with a bovine growth hormone poly-adenylation signal (PA). ITR, inverted terminal repeat; CMV, cytomegalovirus promoter; PCDS, partial coding DNA sequence of CEP290 ; 5′ SS, 5′ splice site. (B) qPCR was performed on HEK293T transfected with a plasmid encoding GFP as a transfection control or the plasmids in (A). TaqMan probes were designed to the junctions indicated: CEP290 exons 26 and 27 (hEx26-hEx27), a region within the 5′ codon optimized partial coding DNA sequence (coPCDS), or the novel junction of 5′ coPCDS and endogenous Homo sapiens exon 27 to signify trans -splicing (coPCDS-hEx27). Significant variation within replicates for coPCDS-hEx27 was present because the Ct was crossed at 35 cycles with either PTM; however, no amplification was observed through 40 cycles in GFP-treated samples. Samples were standardized to β-2-microglobin and normalized to NPA. Error bars are relative quantity minimum and maximum 95% confidence intervals. (C) Agarose gel electrophoresis of one of the replicate reactions from (B). (D) Densitometry analysis of the bands in (C). (E) Sanger sequencing following TOPO-cloning of the PCR product comprising the coPCDS-hEx27 junction visualized in (C). Nucleotide differences between Homo sapiens and codon-optimized CEP290 are noted by asterisks. The junction between the 5′ coPCDS and endogenous exon 27 is marked by a vertical dashed line.
    Figure Legend Snippet: Editing of CEP290 Transcripts Occurs in HEK293T after Transfection with PTMs (A) Illustration of adeno-associated virus genome arrangement of a CEP290 5′ PTM with binding domain 07 (PTM_07) with either no poly-adenylation signal (NPA) or with a bovine growth hormone poly-adenylation signal (PA). ITR, inverted terminal repeat; CMV, cytomegalovirus promoter; PCDS, partial coding DNA sequence of CEP290 ; 5′ SS, 5′ splice site. (B) qPCR was performed on HEK293T transfected with a plasmid encoding GFP as a transfection control or the plasmids in (A). TaqMan probes were designed to the junctions indicated: CEP290 exons 26 and 27 (hEx26-hEx27), a region within the 5′ codon optimized partial coding DNA sequence (coPCDS), or the novel junction of 5′ coPCDS and endogenous Homo sapiens exon 27 to signify trans -splicing (coPCDS-hEx27). Significant variation within replicates for coPCDS-hEx27 was present because the Ct was crossed at 35 cycles with either PTM; however, no amplification was observed through 40 cycles in GFP-treated samples. Samples were standardized to β-2-microglobin and normalized to NPA. Error bars are relative quantity minimum and maximum 95% confidence intervals. (C) Agarose gel electrophoresis of one of the replicate reactions from (B). (D) Densitometry analysis of the bands in (C). (E) Sanger sequencing following TOPO-cloning of the PCR product comprising the coPCDS-hEx27 junction visualized in (C). Nucleotide differences between Homo sapiens and codon-optimized CEP290 are noted by asterisks. The junction between the 5′ coPCDS and endogenous exon 27 is marked by a vertical dashed line.

    Techniques Used: Transfection, Binding Assay, Sequencing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Amplification, Agarose Gel Electrophoresis, Clone Assay, Polymerase Chain Reaction

    Editing of Mini- CEP290 Transcripts Occurs In Vivo following Sub-retinal Injection of 7m8AAV-5′ PTMs (A) Diagram of the CEP290 intron 26 mini-gene. The mini-gene is driven by the murine Rhodopsin promoter (mRho). CEP290 exons 25 and 26 are joined and followed by the complete intron 26-27 and exon 27. An A > G mutation corresponding to c.2991+1655 was included to assess exon X splicing in the model. An amino-terminal Myc and a carboxy-terminal FLAG tag were added to flank the mini-gene. Additionally, an internal ribosomal entry site (IRES) was added to drive expression of EGFP and is terminated by a bovine growth hormone poly-adenylation signal sequence (pA). (B) Immunofluorescence staining of a retinal cross-section from a mini- CEP290 mouse. OS, outer segment. ONL, outer nuclear layer. INL, inner nuclear layer. Scale bar is 50 μm. (C) Illustration of potential splicing outcomes within the mini- CEP290 mouse. Canonical cis- splicing would result in a predicted 24.3-kDa peptide. Alternative cis- splicing with exon X would yield a predicted non-sense media decay transcript encoding a truncated peptide with Myc at 17.1 kDa and no FLAG translation. Trans -splicing with a 5′ PTM would replace the amino-terminal Myc and generate a 124.1-kDa peptide with a FLAG tag. Arrow pairs indicate locations of PCR primers used to detect specific splicing events. (D) Representative western blot images of Myc and FLAG at 24.3 kDa showing OD and OS lanes per animal for each contralateral treatment cohort. (E) Densitometry quantification showing the mean log 10 values of each contralateral treatment cohort. Samples were standardized to α-tubulin. Error bars are SEM. Sample sizes as indicated. Individual animal data is available in Figure S1 A. (F) qPCR from cDNA generated by RNA extracts from whole eyes of mini- CEP290 mice. TaqMan probes were designed to the junctions of Homo sapiens exon 27 and FLAG to detect total expression of the mini-gene (left) to a region within the PCDS to detect total expression of the PTM (center) or to the novel junction of codon-optimized CEP290 PCDS and Homo sapiens CEP290 exon 27 (right). Values from treatment-matched samples were averaged as biological groups, standardized to murine β-2-microglobin and normalized to PTM_NBD. p
    Figure Legend Snippet: Editing of Mini- CEP290 Transcripts Occurs In Vivo following Sub-retinal Injection of 7m8AAV-5′ PTMs (A) Diagram of the CEP290 intron 26 mini-gene. The mini-gene is driven by the murine Rhodopsin promoter (mRho). CEP290 exons 25 and 26 are joined and followed by the complete intron 26-27 and exon 27. An A > G mutation corresponding to c.2991+1655 was included to assess exon X splicing in the model. An amino-terminal Myc and a carboxy-terminal FLAG tag were added to flank the mini-gene. Additionally, an internal ribosomal entry site (IRES) was added to drive expression of EGFP and is terminated by a bovine growth hormone poly-adenylation signal sequence (pA). (B) Immunofluorescence staining of a retinal cross-section from a mini- CEP290 mouse. OS, outer segment. ONL, outer nuclear layer. INL, inner nuclear layer. Scale bar is 50 μm. (C) Illustration of potential splicing outcomes within the mini- CEP290 mouse. Canonical cis- splicing would result in a predicted 24.3-kDa peptide. Alternative cis- splicing with exon X would yield a predicted non-sense media decay transcript encoding a truncated peptide with Myc at 17.1 kDa and no FLAG translation. Trans -splicing with a 5′ PTM would replace the amino-terminal Myc and generate a 124.1-kDa peptide with a FLAG tag. Arrow pairs indicate locations of PCR primers used to detect specific splicing events. (D) Representative western blot images of Myc and FLAG at 24.3 kDa showing OD and OS lanes per animal for each contralateral treatment cohort. (E) Densitometry quantification showing the mean log 10 values of each contralateral treatment cohort. Samples were standardized to α-tubulin. Error bars are SEM. Sample sizes as indicated. Individual animal data is available in Figure S1 A. (F) qPCR from cDNA generated by RNA extracts from whole eyes of mini- CEP290 mice. TaqMan probes were designed to the junctions of Homo sapiens exon 27 and FLAG to detect total expression of the mini-gene (left) to a region within the PCDS to detect total expression of the PTM (center) or to the novel junction of codon-optimized CEP290 PCDS and Homo sapiens CEP290 exon 27 (right). Values from treatment-matched samples were averaged as biological groups, standardized to murine β-2-microglobin and normalized to PTM_NBD. p

    Techniques Used: In Vivo, Injection, Mutagenesis, FLAG-tag, Expressing, Sequencing, Immunofluorescence, Staining, Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction, Generated, Mouse Assay

    4) Product Images from "Uncoupling Antisense-Mediated Silencing and DNA Methylation in the Imprinted Gnas Cluster"

    Article Title: Uncoupling Antisense-Mediated Silencing and DNA Methylation in the Imprinted Gnas Cluster

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001347

    Nesp is fully expressed from the paternal allele in +/ Nespas-T ex1 . (A) The Nespas-T ex1 allele was generated by targeting and is identical to Nesp-T int2 except that the polyadenylation cassette was inserted in the opposite orientation (Ap) to truncate Nespas. The selection genes were deleted from the Ap- neo targeted allele upon germline transmission by Cre-recombinase mediated excision to generate Nespas-T ex1 . (B) Southern analysis of ES cell DNA from wild-type (+/+) and targeted (Ap- neo ) cells. The Ap- neo targeted clones were identified by the presence of an 8.5 kb Nde I fragment detected with the 3′ external probe. Correct targeting at the 5′ end was confirmed by the detection of a 12.9 kb Avr II fragment with the 5′ external probe. (C) RT-PCR assay in newborn brain showing Nespas is truncated. A primer pair, 3′ of the insertion, detected Nespas whereas a primer pair 5′ of the insertion did not detect Nespas . Symbols + and −, refer to reactions carried out in the presence and absence of reverse transcriptase, respectively. As the Nespas PCR products were weak, it was necessary to blot and probe the Nespas RT-PCR products with appropriate genomic probes. Hprt was included as an amplification control. (D) Southern analysis showing promoter methylation at the Nesp DMR is lost in +/ Nespas-T ex1 . Genomic DNA from newborn brain was digested with Eco RI (-), Eco RI and Hpa II (H) or Eco RI and Msp I (M). (E) Biallelic expression of Nesp in +/ Nespas-T ex1 . (E Top Left) RNA blot analysis showing expression of Nesp in poly(A) + RNA from 15.5 dpc embryos. (E Bottom Left) Schematic summary of the transcriptional and methylation status of Nespas and Nesp on the paternal allele of +/ Nespas-T ex1 . (E Right) Bar chart showing the relative level of Nesp expression in newborn brain by RT-qPCR, measured using a TaqMan assay that detects exon 1 spliced onto 2. The reference gene was Gapdh . Error bars (RQmin/RQmax) were based on a 95% confidence level.
    Figure Legend Snippet: Nesp is fully expressed from the paternal allele in +/ Nespas-T ex1 . (A) The Nespas-T ex1 allele was generated by targeting and is identical to Nesp-T int2 except that the polyadenylation cassette was inserted in the opposite orientation (Ap) to truncate Nespas. The selection genes were deleted from the Ap- neo targeted allele upon germline transmission by Cre-recombinase mediated excision to generate Nespas-T ex1 . (B) Southern analysis of ES cell DNA from wild-type (+/+) and targeted (Ap- neo ) cells. The Ap- neo targeted clones were identified by the presence of an 8.5 kb Nde I fragment detected with the 3′ external probe. Correct targeting at the 5′ end was confirmed by the detection of a 12.9 kb Avr II fragment with the 5′ external probe. (C) RT-PCR assay in newborn brain showing Nespas is truncated. A primer pair, 3′ of the insertion, detected Nespas whereas a primer pair 5′ of the insertion did not detect Nespas . Symbols + and −, refer to reactions carried out in the presence and absence of reverse transcriptase, respectively. As the Nespas PCR products were weak, it was necessary to blot and probe the Nespas RT-PCR products with appropriate genomic probes. Hprt was included as an amplification control. (D) Southern analysis showing promoter methylation at the Nesp DMR is lost in +/ Nespas-T ex1 . Genomic DNA from newborn brain was digested with Eco RI (-), Eco RI and Hpa II (H) or Eco RI and Msp I (M). (E) Biallelic expression of Nesp in +/ Nespas-T ex1 . (E Top Left) RNA blot analysis showing expression of Nesp in poly(A) + RNA from 15.5 dpc embryos. (E Bottom Left) Schematic summary of the transcriptional and methylation status of Nespas and Nesp on the paternal allele of +/ Nespas-T ex1 . (E Right) Bar chart showing the relative level of Nesp expression in newborn brain by RT-qPCR, measured using a TaqMan assay that detects exon 1 spliced onto 2. The reference gene was Gapdh . Error bars (RQmin/RQmax) were based on a 95% confidence level.

    Techniques Used: Generated, Selection, Transmission Assay, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Methylation, Expressing, Northern blot, Quantitative RT-PCR, TaqMan Assay

    Maternal transmission of Nesp-T int2 results in downregulation of Nesp when the Nespas DMR is unmethylated. (A) The maternal Nesp transcript is truncated in Nesp-T int2 /+. RNA blot analysis showing expression of Nesp in poly(A) + RNA from 15.5 dpc embryos using the single stranded RNA probe shown in Figure 2C . (B) Southern analysis showing the Nespas - Gnasxl DMR promoter can be unmethylated or methylated on the maternal allele in Nesp-T int2 /+. Genomic DNA from newborn brain was digested with Eco RI (-), Eco RI and Hpa II (H), or Eco RI and Msp I (M) and probed as shown in Figure S1 . (C) Schematic showing the genomic organization of the double heterozygote with Nesp-T int2 maternally inherited and Δ NAS-DMR paternally inherited. The polyadenylation cassette, pA, truncates Nesp on the maternal allele and the ICR is deleted on the paternal allele. (D) RT-PCR showing Nespas is weakly expressed in neonatal brain from the maternally-derived targeted allele when the Nespas DMR is unmethylated. Primers were as described in Figure 2C . + and −, presence and absence of reverse transcriptase, respectively; Hprt , amplification control. (E, Left) Schematic summary of the transcriptional and methylation status of the maternal allele of Nesp and Nespas in Nesp-T int2 /+ (methylated) and Nesp-T int2 /+ (unmethylated) newborn brain. The polyadenylation cassette, pA truncates Nesp on the maternal allele. Row of filled circles, methylated allele; row of open circles, unmethylated allele. (E Right) Bar chart showing the relative level of Nesp expression in newborn brain by RT-qPCR between Nesp-T int2 /+ SD2 littermates that had the Nespas DMR methylated and unmethylated. Nesp levels were measured using the TaqMan assay detecting exon 1 spliced onto exon 2. The level of Nesp was significantly lower in littermates that had the Nespas DMR unmethylated compared with those that had a methylated DMR ( P = 5.4×10 −3 ).
    Figure Legend Snippet: Maternal transmission of Nesp-T int2 results in downregulation of Nesp when the Nespas DMR is unmethylated. (A) The maternal Nesp transcript is truncated in Nesp-T int2 /+. RNA blot analysis showing expression of Nesp in poly(A) + RNA from 15.5 dpc embryos using the single stranded RNA probe shown in Figure 2C . (B) Southern analysis showing the Nespas - Gnasxl DMR promoter can be unmethylated or methylated on the maternal allele in Nesp-T int2 /+. Genomic DNA from newborn brain was digested with Eco RI (-), Eco RI and Hpa II (H), or Eco RI and Msp I (M) and probed as shown in Figure S1 . (C) Schematic showing the genomic organization of the double heterozygote with Nesp-T int2 maternally inherited and Δ NAS-DMR paternally inherited. The polyadenylation cassette, pA, truncates Nesp on the maternal allele and the ICR is deleted on the paternal allele. (D) RT-PCR showing Nespas is weakly expressed in neonatal brain from the maternally-derived targeted allele when the Nespas DMR is unmethylated. Primers were as described in Figure 2C . + and −, presence and absence of reverse transcriptase, respectively; Hprt , amplification control. (E, Left) Schematic summary of the transcriptional and methylation status of the maternal allele of Nesp and Nespas in Nesp-T int2 /+ (methylated) and Nesp-T int2 /+ (unmethylated) newborn brain. The polyadenylation cassette, pA truncates Nesp on the maternal allele. Row of filled circles, methylated allele; row of open circles, unmethylated allele. (E Right) Bar chart showing the relative level of Nesp expression in newborn brain by RT-qPCR between Nesp-T int2 /+ SD2 littermates that had the Nespas DMR methylated and unmethylated. Nesp levels were measured using the TaqMan assay detecting exon 1 spliced onto exon 2. The level of Nesp was significantly lower in littermates that had the Nespas DMR unmethylated compared with those that had a methylated DMR ( P = 5.4×10 −3 ).

    Techniques Used: Transmission Assay, Northern blot, Expressing, Methylation, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Amplification, Quantitative RT-PCR, TaqMan Assay

    The targeted allele, Nesp-T int2 , is a Nespas hypomorph when paternally inherited. (A) Overview of the mouse Gnas locus showing enlargement of the Nespas region and the site of insertion of the rabbit β-globin polyadenylation cassette in Nespas exon 1. The targeting vector shows the location of a 1.2 kb fragment (pA) from the rabbit β-globin gene to truncate Nesp , and the selection genes were flanked by loxP sites (open triangles). The selection genes were deleted from the pA- neo targeted allele upon male germline transmission by Cre-recombinase mediated excision to generate Nesp-T int2 . The DMRs are shown by the presence of filled circles on the methylated allele. A, Avr II; N, Nde I; Sp, Spe I; Sw, Swa I; P, Nespas promoter; ICR, Imprinting Control Region, the extent of which is defined by the deletion in the Δ NAS-DMR allele [8] . (B) Southern analysis of ES cell DNA from wild-type (+/+) and targeted (pA- neo ) cells. The pA- neo targeted clones were identified by the presence of an 8.5 kb Nde I fragment detected with the 3′ external probe. Correct targeting at the 5′ end was confirmed by the detection of a 12.9 kb Avr II fragment with the 5′ external probe. (C) Schematic of the Nesp - Nespas region showing overlapping transcription on the sense and antisense strands and the position of RT-PCR primers, a single stranded RNA probe and TaqMan assay for detecting the primary Nespas transcript. (D) RT-PCR analysis of Nespas in neonatal brain after paternal transmission of Nesp-T int2 (+/ Nesp-T int2 ) using primers F6 and R2 (Figure 2C; Table S1 ). +/Δ NAS-DMR , mice with paternal deletion of the ICR [8] ; + and −, presence and absence of reverse transcriptase, respectively; Hprt , amplification control. (E) RNA blot analysis showing expression of Nespas in poly(A) + RNA from 15.5 dpc embryos using the single stranded RNA probe shown in Figure 2C. (F) Bar chart showing the relative level of Nespas expression in newborn brain by RT-qPCR using a TaqMan assay within intron 4 (Figure 2C). The reference gene was Gapdh . Error bars (RQmin/RQmax) were based on a 95% confidence level.
    Figure Legend Snippet: The targeted allele, Nesp-T int2 , is a Nespas hypomorph when paternally inherited. (A) Overview of the mouse Gnas locus showing enlargement of the Nespas region and the site of insertion of the rabbit β-globin polyadenylation cassette in Nespas exon 1. The targeting vector shows the location of a 1.2 kb fragment (pA) from the rabbit β-globin gene to truncate Nesp , and the selection genes were flanked by loxP sites (open triangles). The selection genes were deleted from the pA- neo targeted allele upon male germline transmission by Cre-recombinase mediated excision to generate Nesp-T int2 . The DMRs are shown by the presence of filled circles on the methylated allele. A, Avr II; N, Nde I; Sp, Spe I; Sw, Swa I; P, Nespas promoter; ICR, Imprinting Control Region, the extent of which is defined by the deletion in the Δ NAS-DMR allele [8] . (B) Southern analysis of ES cell DNA from wild-type (+/+) and targeted (pA- neo ) cells. The pA- neo targeted clones were identified by the presence of an 8.5 kb Nde I fragment detected with the 3′ external probe. Correct targeting at the 5′ end was confirmed by the detection of a 12.9 kb Avr II fragment with the 5′ external probe. (C) Schematic of the Nesp - Nespas region showing overlapping transcription on the sense and antisense strands and the position of RT-PCR primers, a single stranded RNA probe and TaqMan assay for detecting the primary Nespas transcript. (D) RT-PCR analysis of Nespas in neonatal brain after paternal transmission of Nesp-T int2 (+/ Nesp-T int2 ) using primers F6 and R2 (Figure 2C; Table S1 ). +/Δ NAS-DMR , mice with paternal deletion of the ICR [8] ; + and −, presence and absence of reverse transcriptase, respectively; Hprt , amplification control. (E) RNA blot analysis showing expression of Nespas in poly(A) + RNA from 15.5 dpc embryos using the single stranded RNA probe shown in Figure 2C. (F) Bar chart showing the relative level of Nespas expression in newborn brain by RT-qPCR using a TaqMan assay within intron 4 (Figure 2C). The reference gene was Gapdh . Error bars (RQmin/RQmax) were based on a 95% confidence level.

    Techniques Used: Plasmid Preparation, Selection, Transmission Assay, Methylation, Clone Assay, Reverse Transcription Polymerase Chain Reaction, TaqMan Assay, Mouse Assay, Amplification, Northern blot, Expressing, Quantitative RT-PCR

    5) Product Images from "Defining the Transcriptional and Cellular Landscape of Type 1 Diabetes in the NOD Mouse"

    Article Title: Defining the Transcriptional and Cellular Landscape of Type 1 Diabetes in the NOD Mouse

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0059701

    Quantitative RT-PCR validation of microarray data. ( A ) Quantitative RT-PCR was performed using SYBR green detection for the indicated genes. Bars show the mean (log 2 ) +/− S.E.M. of at least three independent experimental replicates from 3–6 biological replicates per group. All data is represented relative to the expression of actin (ΔC t ). In order to facilitate visualization on a log 2 scale, values were transformed as indicated on the y-axis label. ( B ) Microarray results for the same genes interrogated in ( A ). ( C ) Taqman qPCR quantification of pan-IFNα, Ifnb1 , or Ifng throughout diabetogenesis. Bars represent the mean of the normalized probe intensity +/− S.E.M. of 3–6 biological replicates per group. Asterisks indicate statistical significance (P
    Figure Legend Snippet: Quantitative RT-PCR validation of microarray data. ( A ) Quantitative RT-PCR was performed using SYBR green detection for the indicated genes. Bars show the mean (log 2 ) +/− S.E.M. of at least three independent experimental replicates from 3–6 biological replicates per group. All data is represented relative to the expression of actin (ΔC t ). In order to facilitate visualization on a log 2 scale, values were transformed as indicated on the y-axis label. ( B ) Microarray results for the same genes interrogated in ( A ). ( C ) Taqman qPCR quantification of pan-IFNα, Ifnb1 , or Ifng throughout diabetogenesis. Bars represent the mean of the normalized probe intensity +/− S.E.M. of 3–6 biological replicates per group. Asterisks indicate statistical significance (P

    Techniques Used: Quantitative RT-PCR, Microarray, SYBR Green Assay, Expressing, Transformation Assay, Real-time Polymerase Chain Reaction

    6) Product Images from "Cidofovir selectivity is based on the different response of normal and cancer cells to DNA damage"

    Article Title: Cidofovir selectivity is based on the different response of normal and cancer cells to DNA damage

    Journal: BMC Medical Genomics

    doi: 10.1186/1755-8794-6-18

    Gene expression evaluated by real-time RT-PCR. ( A ) The TaqMan® Fast Universal PCR Master Mix and TaqMan® Gene Expression Assays from Applied Biosystems were employed to determine expression of the following genes (with assay ID’s): AOX1 (Hs00154079_m1), DHRS2 (Hs01061576_m1), HIST1H2AC (Hs00374312_s1), ICAM4 (Hs00169941_m1), MAP2K6 (Hs00992389_m1), OSMR (Hs00384276_m1), TP53 (Hs99999147_m1), and RB1 (Hs01078075_m1) in SiHa, HeLa, HaCaT, and PHKs following CDV treatment for 72 h. White bars represent the relative expression values of the microarray data. Black bars represent average RT-PCR values (±SD), relative to untreated cells and normalized against β–actin, from three independent samples. ( B ) Relative expression levels of HPV16 E6 and E7 in SiHa cells following treatment with CDV for different times, relative to untreated SiHa cells and normalized against β–actin. The bars show average values (±SD) of at least three independent experiments. An absolute 2-fold change difference was considered as biologically significant and is indicated by dashed lines in the figures.
    Figure Legend Snippet: Gene expression evaluated by real-time RT-PCR. ( A ) The TaqMan® Fast Universal PCR Master Mix and TaqMan® Gene Expression Assays from Applied Biosystems were employed to determine expression of the following genes (with assay ID’s): AOX1 (Hs00154079_m1), DHRS2 (Hs01061576_m1), HIST1H2AC (Hs00374312_s1), ICAM4 (Hs00169941_m1), MAP2K6 (Hs00992389_m1), OSMR (Hs00384276_m1), TP53 (Hs99999147_m1), and RB1 (Hs01078075_m1) in SiHa, HeLa, HaCaT, and PHKs following CDV treatment for 72 h. White bars represent the relative expression values of the microarray data. Black bars represent average RT-PCR values (±SD), relative to untreated cells and normalized against β–actin, from three independent samples. ( B ) Relative expression levels of HPV16 E6 and E7 in SiHa cells following treatment with CDV for different times, relative to untreated SiHa cells and normalized against β–actin. The bars show average values (±SD) of at least three independent experiments. An absolute 2-fold change difference was considered as biologically significant and is indicated by dashed lines in the figures.

    Techniques Used: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Microarray, Reverse Transcription Polymerase Chain Reaction

    7) Product Images from "Optimized Surface Markers for the Prospective Isolation of High-Quality hiPSCs using Flow Cytometry Selection"

    Article Title: Optimized Surface Markers for the Prospective Isolation of High-Quality hiPSCs using Flow Cytometry Selection

    Journal: Scientific Reports

    doi: 10.1038/srep01179

    hiPSC selection by sorting SSEA4 + TRA-1-81 + CD30 + cells yields pluripotent feeder-free hiPSC lines with stable genome and tri-lineage differentiation potential. Four weeks post initiation of reprogramming of FTc91 human fibroblasts, hiPSCs were separated by FACS and using the SSEA4, TRA-1-81, and CD30 markers as described above. Three iPSC lines FTi115, FTi116, and FTi117 were established and characterized. (a) Flow cytometry analyses of indicated hiPSCs. (b) Indicated iPSC lines were immunostained for expression of NANOG (green) and OCT4 (red). Nuclei were stained with Hoechst dye (blue). Scale bar is 200 μm. (c) Nanog+ and Oct4+ FTi117 iPSCs were quanitified by intracellular flow cytometry. (d), (e) qRT-PCR analysis of gene expression of pluripotency markers (d) and transgene (e) in indicated hiPSC lines. The lentivirus used for reprogramming expressed the transgenes as a single polycistronic cassette. Transgene levels were measured by a TaqMan primer-probe set within the viral WPRE element. Error bars represent standard deviation of duplicates. The asterisks denote corresponding p-values (*
    Figure Legend Snippet: hiPSC selection by sorting SSEA4 + TRA-1-81 + CD30 + cells yields pluripotent feeder-free hiPSC lines with stable genome and tri-lineage differentiation potential. Four weeks post initiation of reprogramming of FTc91 human fibroblasts, hiPSCs were separated by FACS and using the SSEA4, TRA-1-81, and CD30 markers as described above. Three iPSC lines FTi115, FTi116, and FTi117 were established and characterized. (a) Flow cytometry analyses of indicated hiPSCs. (b) Indicated iPSC lines were immunostained for expression of NANOG (green) and OCT4 (red). Nuclei were stained with Hoechst dye (blue). Scale bar is 200 μm. (c) Nanog+ and Oct4+ FTi117 iPSCs were quanitified by intracellular flow cytometry. (d), (e) qRT-PCR analysis of gene expression of pluripotency markers (d) and transgene (e) in indicated hiPSC lines. The lentivirus used for reprogramming expressed the transgenes as a single polycistronic cassette. Transgene levels were measured by a TaqMan primer-probe set within the viral WPRE element. Error bars represent standard deviation of duplicates. The asterisks denote corresponding p-values (*

    Techniques Used: Selection, FACS, Flow Cytometry, Cytometry, Expressing, Staining, Quantitative RT-PCR, Standard Deviation

    8) Product Images from "Region-Specific Differences in Amyloid Precursor Protein Expression in the Mouse Hippocampus"

    Article Title: Region-Specific Differences in Amyloid Precursor Protein Expression in the Mouse Hippocampus

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2016.00134

    Quantitative App mRNA expression in hippocampal subregions. (A,B) LMD of hippocampal subregions, i.e., CA1 pcl and gcl of the DG. Representative section of the dorsal hippocampus (coronal plane, cresyl violet staining) before (A) and after (B) LMD is shown. Scale bar: 250 μm. (C) RNA integrity analysis of total RNA isolated from the dissected gcl (red) and from pcl (blue) demonstrating highly intact RNA (RIN-values: ~9; Agilent 2100 Bioanalyzer). (D,E) Average expression stability values ( M ) and evaluation of the optimum number of candidate reference genes for CA1 pcl and for DG gcl according to geNorm software. Pairwise variation (V) of candidate reference genes indicates that the use of the two most stable genes is sufficient to obtain an accurate normalization index for quantitative PCR (qPCR) analysis. (F) A significantly higher gene expression for App (1.5- to 1.7-fold) was detected in CA1 pcl relative to DG gcl using two different App -specific TaqMan assays ( A : Mm_01344172_m1, B : Mm_00431830_m1) after normalization to a reference gene index calculated by geNorm. (G,H) Gene expression stability values ( S ) and accumulated SD analysis using NormFinder. The minimal number of reference genes required for effective normalization is highlighted. (I) Comparable to the results obtained by geNorm algorithm, a significantly higher App expression (1.6- to 1.7-fold) was detected in CA1 pcl relative to DG gcl after normalization to the reference gene index estimated by NormFinder. Data ( N = 5–6 mice) were tested for statistical significance using one-way ANOVA followed by Bonferroni post hoc test to correct for multiple comparisons, * p ≤ 0.05.
    Figure Legend Snippet: Quantitative App mRNA expression in hippocampal subregions. (A,B) LMD of hippocampal subregions, i.e., CA1 pcl and gcl of the DG. Representative section of the dorsal hippocampus (coronal plane, cresyl violet staining) before (A) and after (B) LMD is shown. Scale bar: 250 μm. (C) RNA integrity analysis of total RNA isolated from the dissected gcl (red) and from pcl (blue) demonstrating highly intact RNA (RIN-values: ~9; Agilent 2100 Bioanalyzer). (D,E) Average expression stability values ( M ) and evaluation of the optimum number of candidate reference genes for CA1 pcl and for DG gcl according to geNorm software. Pairwise variation (V) of candidate reference genes indicates that the use of the two most stable genes is sufficient to obtain an accurate normalization index for quantitative PCR (qPCR) analysis. (F) A significantly higher gene expression for App (1.5- to 1.7-fold) was detected in CA1 pcl relative to DG gcl using two different App -specific TaqMan assays ( A : Mm_01344172_m1, B : Mm_00431830_m1) after normalization to a reference gene index calculated by geNorm. (G,H) Gene expression stability values ( S ) and accumulated SD analysis using NormFinder. The minimal number of reference genes required for effective normalization is highlighted. (I) Comparable to the results obtained by geNorm algorithm, a significantly higher App expression (1.6- to 1.7-fold) was detected in CA1 pcl relative to DG gcl after normalization to the reference gene index estimated by NormFinder. Data ( N = 5–6 mice) were tested for statistical significance using one-way ANOVA followed by Bonferroni post hoc test to correct for multiple comparisons, * p ≤ 0.05.

    Techniques Used: Expressing, Laser Capture Microdissection, Staining, Isolation, Software, Real-time Polymerase Chain Reaction, Mouse Assay

    9) Product Images from "Aging and calorie restriction regulate the expression of miR-125a-5p and its target genes Stat3, Casp2 and Stard13"

    Article Title: Aging and calorie restriction regulate the expression of miR-125a-5p and its target genes Stat3, Casp2 and Stard13

    Journal: Aging (Albany NY)

    doi: 10.18632/aging.101270

    30% CR affected the MicroRNA expression in mouse liver The expression of indicated miRNAs was assayed using ( A ) microarray hybridization and ( B ) Taqman PCR assay in the liver of AL (Open bars) and CR mice (Black bars). * - statistically significant difference (p
    Figure Legend Snippet: 30% CR affected the MicroRNA expression in mouse liver The expression of indicated miRNAs was assayed using ( A ) microarray hybridization and ( B ) Taqman PCR assay in the liver of AL (Open bars) and CR mice (Black bars). * - statistically significant difference (p

    Techniques Used: Expressing, Microarray, Hybridization, Polymerase Chain Reaction, Mouse Assay

    10) Product Images from "RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes"

    Article Title: RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00775

    Design and execution of the small-hairpin (sh)RNA screens for murine nuclear cofactors of IL-1 signaling. For screen I, 4–5 shRNAs directed at up to four different nuclear targets per 48-well plate were transfected in duplicates as shown. In screen II, 4–5 shRNAs per nuclear target were pooled and transfected into a single well resulting in screening of 20 nuclear targets per plate (not shown). In both screens, empty vector (pLKO.1) with no insert or an insert encoding a scrambled shRNA sequence (pLKO.1-scr.) were used as controls on each of the plates. Cells transfected with pLKO.1 encoding a GFP cDNA were used to monitor transfection efficiency on each individual plate by fluorescence microscopy (left image) and by phase contrast plus fluorescence microscopy (right image) as shown by the insets. The scale bar is 100 µm. For each screen, 3.5 × 10 4 cells were seeded per well. One day later, 270 ng of DNA were transfected using Lipofectamine LTX plus reagent ® . Cells were selected for 48 h in 1 µg/ml puromycin. Then, half of the cells on each plate were left untreated. The other half was stimulated for 3 h (screen I) or 1 h (screen II) with IL-1α (10 ng/ml). Thereafter, cells were lysed, and RT reactions and preamplifications of cDNAs were performed using the PreAmp Cells-to-Ct TM Kit and gene specific primers for the IL-1-inducible target gene Cxcl2 and the two “housekeeping” genes ActB (screen I only) and Ube2l3 . Finally, preamplified PCR products were subjected to quantitative (q)PCR using Taqman assays and mRNA expressions levels were quantified based on their cycle threshold (ct) value using an ABI7500 instrument. Ct values were used for all further calculations.
    Figure Legend Snippet: Design and execution of the small-hairpin (sh)RNA screens for murine nuclear cofactors of IL-1 signaling. For screen I, 4–5 shRNAs directed at up to four different nuclear targets per 48-well plate were transfected in duplicates as shown. In screen II, 4–5 shRNAs per nuclear target were pooled and transfected into a single well resulting in screening of 20 nuclear targets per plate (not shown). In both screens, empty vector (pLKO.1) with no insert or an insert encoding a scrambled shRNA sequence (pLKO.1-scr.) were used as controls on each of the plates. Cells transfected with pLKO.1 encoding a GFP cDNA were used to monitor transfection efficiency on each individual plate by fluorescence microscopy (left image) and by phase contrast plus fluorescence microscopy (right image) as shown by the insets. The scale bar is 100 µm. For each screen, 3.5 × 10 4 cells were seeded per well. One day later, 270 ng of DNA were transfected using Lipofectamine LTX plus reagent ® . Cells were selected for 48 h in 1 µg/ml puromycin. Then, half of the cells on each plate were left untreated. The other half was stimulated for 3 h (screen I) or 1 h (screen II) with IL-1α (10 ng/ml). Thereafter, cells were lysed, and RT reactions and preamplifications of cDNAs were performed using the PreAmp Cells-to-Ct TM Kit and gene specific primers for the IL-1-inducible target gene Cxcl2 and the two “housekeeping” genes ActB (screen I only) and Ube2l3 . Finally, preamplified PCR products were subjected to quantitative (q)PCR using Taqman assays and mRNA expressions levels were quantified based on their cycle threshold (ct) value using an ABI7500 instrument. Ct values were used for all further calculations.

    Techniques Used: Transfection, Plasmid Preparation, shRNA, Sequencing, Fluorescence, Microscopy, Polymerase Chain Reaction

    11) Product Images from "Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes"

    Article Title: Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl416

    The C T value as a function of annealing/elongation time is shown in red. This demonstrated that the Taqman PCR system can be run with an annealing/elongation step of 10 s. Even with this step as short as 6.5 s, the PCR performance is not significantly affected. This PCR system is then capable of running a complete 50 cycle PCR protocol in 7 min and 25 s inclusive of a 20 s hot start.
    Figure Legend Snippet: The C T value as a function of annealing/elongation time is shown in red. This demonstrated that the Taqman PCR system can be run with an annealing/elongation step of 10 s. Even with this step as short as 6.5 s, the PCR performance is not significantly affected. This PCR system is then capable of running a complete 50 cycle PCR protocol in 7 min and 25 s inclusive of a 20 s hot start.

    Techniques Used: Polymerase Chain Reaction

    12) Product Images from "Bortezomib prevents cytarabine resistance in MCL, which is characterized by down-regulation of dCK and up-regulation of SPIB resulting in high NF-κB activity"

    Article Title: Bortezomib prevents cytarabine resistance in MCL, which is characterized by down-regulation of dCK and up-regulation of SPIB resulting in high NF-κB activity

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4346-1

    Effects of SPIB knock-down on resistant cells. a Conformation of partial silencing of SPIB on gene level. The relative mRNA expression of SPIB was assessed in the cells using TaqMan probe based RT-PCR and revealed a knock of gene expression with 50% compared to un-knocked control cells. The data are normalised to the S18 reference gene. b Protein levels of SPIB measured with western blot 48 h after transfection with siRNA. GAPDH was used as loading control. Representative figure of three biological replicates. c Normalised proliferation for Z138 cells grown in cytarabine containing medium, measured by incorporation of [methyl-14C]-thymidine 72 h after knock of SPIB using siRNA. Each data point represents a mean value of triplicates and error bars show SD. * = p
    Figure Legend Snippet: Effects of SPIB knock-down on resistant cells. a Conformation of partial silencing of SPIB on gene level. The relative mRNA expression of SPIB was assessed in the cells using TaqMan probe based RT-PCR and revealed a knock of gene expression with 50% compared to un-knocked control cells. The data are normalised to the S18 reference gene. b Protein levels of SPIB measured with western blot 48 h after transfection with siRNA. GAPDH was used as loading control. Representative figure of three biological replicates. c Normalised proliferation for Z138 cells grown in cytarabine containing medium, measured by incorporation of [methyl-14C]-thymidine 72 h after knock of SPIB using siRNA. Each data point represents a mean value of triplicates and error bars show SD. * = p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection

    Verification of dCK mRNA expression in gene microarray samples. The relative mRNA expression of dCK was assessed in the different Z138 subclones using TaqMan probe based RT-PCR and revealed down-regulation in both Z138-CytES and Z138-CytR cells compared to Z138-CytNS cells. The data are normalised to the S18 reference gene, and the reference sample, replicate A of the Z138-CytNS for each run. Bars represent relative quantity ± SEM of three technical replicates. * = p
    Figure Legend Snippet: Verification of dCK mRNA expression in gene microarray samples. The relative mRNA expression of dCK was assessed in the different Z138 subclones using TaqMan probe based RT-PCR and revealed down-regulation in both Z138-CytES and Z138-CytR cells compared to Z138-CytNS cells. The data are normalised to the S18 reference gene, and the reference sample, replicate A of the Z138-CytNS for each run. Bars represent relative quantity ± SEM of three technical replicates. * = p

    Techniques Used: Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction

    13) Product Images from "PPARγ Expression Is Diminished in Macrophages of Recurrent Miscarriage Placentas"

    Article Title: PPARγ Expression Is Diminished in Macrophages of Recurrent Miscarriage Placentas

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19071872

    Results of PPARγ mRNA expression analysis with TaqMan RT-PCR from trophoblastic tissue. PPARγ mRNA expression was significantly downregulated in the miscarriage groups (SM, 15 cases, p = 0.01) and RM, 16 cases, p = 0.004)) compared to the healthy controls (15 cases). This bar graph shows the mean of relative PPARγ expression; therefore, the presentation of error bars is not appropriate.
    Figure Legend Snippet: Results of PPARγ mRNA expression analysis with TaqMan RT-PCR from trophoblastic tissue. PPARγ mRNA expression was significantly downregulated in the miscarriage groups (SM, 15 cases, p = 0.01) and RM, 16 cases, p = 0.004)) compared to the healthy controls (15 cases). This bar graph shows the mean of relative PPARγ expression; therefore, the presentation of error bars is not appropriate.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

    14) Product Images from "Human Cytomegalovirus Gene Expression in Long-Term Infected Glioma Stem Cells"

    Article Title: Human Cytomegalovirus Gene Expression in Long-Term Infected Glioma Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116178

    HCMV gene expression is detectable at 15 weeks p.i only in 387 GSC cells. A. 387 GSC cells infected with AD169 virus (MOI = 2) were analyzed for viral gene expression with SYBR Green RT-PCR at 72 hours, 3 weeks and every 2 weeks thereafter up to 15 weeks. B. Taqman validation of a subset of viral genes of AD169-infected 387 cells at 11 weeks p.i. Relative expression levels were obtained by normalizing viral gene expression with expression of cellular gene Rab14. Each sample was run in triplicate, bars represent S.D. C. Comparison of viral gene expression of AD169-infected glioma stem-like cells and cell lines at 72 hours and 5 weeks p.i. 3832 GSC cells were infected with AD169 virus (MOI = 2) and analyzed for viral gene expression with SYBR Green RT-PCR at 72 hours and 5 weeks p.i. These results are displayed alongside 72 hr and 5 week p.i. data for AD169-infected 387, U87, and T98G from Figures 1D–E and Figure 2A .
    Figure Legend Snippet: HCMV gene expression is detectable at 15 weeks p.i only in 387 GSC cells. A. 387 GSC cells infected with AD169 virus (MOI = 2) were analyzed for viral gene expression with SYBR Green RT-PCR at 72 hours, 3 weeks and every 2 weeks thereafter up to 15 weeks. B. Taqman validation of a subset of viral genes of AD169-infected 387 cells at 11 weeks p.i. Relative expression levels were obtained by normalizing viral gene expression with expression of cellular gene Rab14. Each sample was run in triplicate, bars represent S.D. C. Comparison of viral gene expression of AD169-infected glioma stem-like cells and cell lines at 72 hours and 5 weeks p.i. 3832 GSC cells were infected with AD169 virus (MOI = 2) and analyzed for viral gene expression with SYBR Green RT-PCR at 72 hours and 5 weeks p.i. These results are displayed alongside 72 hr and 5 week p.i. data for AD169-infected 387, U87, and T98G from Figures 1D–E and Figure 2A .

    Techniques Used: Expressing, Infection, SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction

    15) Product Images from "Increases in transient receptor potential vanilloid-1 mRNA and protein in primary afferent neurons stimulated by protein kinase C and their possible role in neurogenic inflammation"

    Article Title: Increases in transient receptor potential vanilloid-1 mRNA and protein in primary afferent neurons stimulated by protein kinase C and their possible role in neurogenic inflammation

    Journal:

    doi: 10.1002/jnr.21844

    CAP-evoked expressions of TRPV 1 and CGRP mRNA ( A and B ) and the effects of a TPRV 1 antagonist and a PKC inhibitor ( C and D ). Relative quantification (RQ) of TRPV 1 and CGRP mRNA level was done by TaqMan® Fast Universal PCR Master Mix and calculated
    Figure Legend Snippet: CAP-evoked expressions of TRPV 1 and CGRP mRNA ( A and B ) and the effects of a TPRV 1 antagonist and a PKC inhibitor ( C and D ). Relative quantification (RQ) of TRPV 1 and CGRP mRNA level was done by TaqMan® Fast Universal PCR Master Mix and calculated

    Techniques Used: Polymerase Chain Reaction

    16) Product Images from "Chronic p27Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor"

    Article Title: Chronic p27Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34475-8

    Expression of GR in clinical lung adenocarcinoma and cell line models. A lung adenocarcinoma tissue microarray was probed by immunohistochemistry for expression of GR and the expression levels were scored in the malignant cells by intensity of staining as well as percent of cells that stained positive (Panel A). Representative images showing the different intensities of staining for GR are shown (Panel B). Whole cell lysates were extracted from H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells and GR expression was visualized by western blot using GAPDH as the loading control (Panel C). RNA was also extracted from the same cell lines and the levels of GR mRNA were measured by real time RT-PCR using TaqMan probes and plotted on a linear scale (Panel D). GR mRNA was measured by quantitative RT-PCR using SYBR Green in a cDNA microarray of clinical lung adenocarcinoma tumors; the relative levels of GR mRNA in H1299 and H1299GR Clone 4 cells were simultaneously measured using SYBR Green and the data plotted on a log scale (dashed lines) (Panel E). Panel D: *P, 0.002; **P, 0.001; ***P, 0.003.
    Figure Legend Snippet: Expression of GR in clinical lung adenocarcinoma and cell line models. A lung adenocarcinoma tissue microarray was probed by immunohistochemistry for expression of GR and the expression levels were scored in the malignant cells by intensity of staining as well as percent of cells that stained positive (Panel A). Representative images showing the different intensities of staining for GR are shown (Panel B). Whole cell lysates were extracted from H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells and GR expression was visualized by western blot using GAPDH as the loading control (Panel C). RNA was also extracted from the same cell lines and the levels of GR mRNA were measured by real time RT-PCR using TaqMan probes and plotted on a linear scale (Panel D). GR mRNA was measured by quantitative RT-PCR using SYBR Green in a cDNA microarray of clinical lung adenocarcinoma tumors; the relative levels of GR mRNA in H1299 and H1299GR Clone 4 cells were simultaneously measured using SYBR Green and the data plotted on a log scale (dashed lines) (Panel E). Panel D: *P, 0.002; **P, 0.001; ***P, 0.003.

    Techniques Used: Expressing, Microarray, Immunohistochemistry, Staining, Western Blot, Quantitative RT-PCR, SYBR Green Assay

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    Amplification:

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    Synthesized:

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    Isolation:

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    Article Snippet: For RT-qPCR analysis, three different reference genes and two target genes were selected for evaluation of their expression profile using exon-spanning TaqMan™ gene expression assays (Applied Biosystems): polymerase (RNA) II Subunit A (Polr2a ), hypoxanthine-guanine phosphoribosyltransferase (Hprt ), glyceraldehyde 3-phosphate dehydrogenase (Gapdh ), glycyl-tRNA synthetase (Gars ) and histone deacetylase 6 (Hdac6 ). .. A master mix for each PCR run was prepared containing TaqMan™ Fast Universal PCR Master Mix (2×, no AmpErase UNG, Applied Biosystems) and 1/20 diluted cDNA was added.

    Size-exclusion Chromatography:

    Article Title: The effects of substance P and acetylcholine on human tenocyte proliferation converge mechanistically via TGF-β1
    Article Snippet: Quantitative PCR was performed using the TaqMan Fast Universal PCR Master Mix (Applied Biosystems, code: 4352042) by splitting the starting volume of 25μl into two 10μl volumes in each level for technical duplicate. .. Quantitative PCR was performed using the TaqMan Fast Universal PCR Master Mix (Applied Biosystems, code: 4352042) by splitting the starting volume of 25μl into two 10μl volumes in each level for technical duplicate.

    Quantitative RT-PCR:

    Article Title: Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infectionSpatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy
    Article Snippet: Paragraph title: Quantitative RT-PCR ... Reverse transcription (RT) was performed using the TaqMan Reverse Transcription Reagents kit (Life Technologies, N8080234) with oligo(dT) primers. cDNA samples were analyzed in triplicate using TaqMan Fast Universal PCR Master Mix (Life Technologies, 4352042) with the following TaqMan gene specific primer-(FAM/MGB) probe mixes (Life Technologies): PML (assay ID Hs00231241_m1), IFI16 (assay ID Hs00986757_m1), ATRX (assay ID HS00997529_m1), Mx1 (assay ID HS00895608_m1) ISG15 (assay ID HS01921425_s1), ISG54 (assay ID Hs01922738_s1), or GAPDH (4333764F) on a 7500 Fast Real time PCR system (Applied Biosystems).

    Article Title: HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth disease
    Article Snippet: For RT-qPCR analysis, three different reference genes and two target genes were selected for evaluation of their expression profile using exon-spanning TaqMan™ gene expression assays (Applied Biosystems): polymerase (RNA) II Subunit A (Polr2a ), hypoxanthine-guanine phosphoribosyltransferase (Hprt ), glyceraldehyde 3-phosphate dehydrogenase (Gapdh ), glycyl-tRNA synthetase (Gars ) and histone deacetylase 6 (Hdac6 ). .. A master mix for each PCR run was prepared containing TaqMan™ Fast Universal PCR Master Mix (2×, no AmpErase UNG, Applied Biosystems) and 1/20 diluted cDNA was added.

    Adsorption:

    Article Title: Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infectionSpatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy
    Article Snippet: Reverse transcription (RT) was performed using the TaqMan Reverse Transcription Reagents kit (Life Technologies, N8080234) with oligo(dT) primers. cDNA samples were analyzed in triplicate using TaqMan Fast Universal PCR Master Mix (Life Technologies, 4352042) with the following TaqMan gene specific primer-(FAM/MGB) probe mixes (Life Technologies): PML (assay ID Hs00231241_m1), IFI16 (assay ID Hs00986757_m1), ATRX (assay ID HS00997529_m1), Mx1 (assay ID HS00895608_m1) ISG15 (assay ID HS01921425_s1), ISG54 (assay ID Hs01922738_s1), or GAPDH (4333764F) on a 7500 Fast Real time PCR system (Applied Biosystems). .. Reverse transcription (RT) was performed using the TaqMan Reverse Transcription Reagents kit (Life Technologies, N8080234) with oligo(dT) primers. cDNA samples were analyzed in triplicate using TaqMan Fast Universal PCR Master Mix (Life Technologies, 4352042) with the following TaqMan gene specific primer-(FAM/MGB) probe mixes (Life Technologies): PML (assay ID Hs00231241_m1), IFI16 (assay ID Hs00986757_m1), ATRX (assay ID HS00997529_m1), Mx1 (assay ID HS00895608_m1) ISG15 (assay ID HS01921425_s1), ISG54 (assay ID Hs01922738_s1), or GAPDH (4333764F) on a 7500 Fast Real time PCR system (Applied Biosystems).

    SYBR Green Assay:

    Article Title: Unusual Five Copies and Dual Forms of nrdB in “Candidatus Liberibacter asiaticus”: Biological Implications and PCR Detection Application
    Article Snippet: For SYBR Green real-time PCR, the reaction mixture contained 10 μl of iQ™ SYBR® Green Supermix (Bio-Rad) or Fast SYBR® Green Master Mix (Applied Biosystems) or Bestar® qPCR Master Mix (DBI® Bioscience), 1 μl of DNA template (~25 ng), 0.5 μl of each forward and reverse primer (10 μM) in a final volume of 20 μl with the following procedure: 95 °C for 3 min (MJ and CFX) or 95 °C for 20 s (ABI), followed by 40 cycles at 95 °C for 10 s (MJ) or 95 °C for 3 s (ABI) or 95 °C for 10 s (CFX, Bio-Rad) and 60 °C for 30 s (MJ and CFX) or 60 °C for 3 s (ABI). .. For TaqMan® real-time PCR, the reaction mixture contained 10 μl of TaqMan® Fast Universal PCR Master Mix (2X) (Applied Biosystems), 1 μl of DNA template (~25 ng), 0.2 μl of TaqMan® probe (5 μM), 0.4 μl of each forward and reverse primer (10 μM) in a final volume of 20 μl with the following procedure: 50 °C for 2 min, then 95 °C for 20 s, followed by 40 cycles at 95 °C for 3 s and 60 °C for 30 s. The fluorescence signal was captured at the end of each 60 °C step.

    Concentration Assay:

    Article Title: Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infectionSpatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy
    Article Snippet: Reverse transcription (RT) was performed using the TaqMan Reverse Transcription Reagents kit (Life Technologies, N8080234) with oligo(dT) primers. cDNA samples were analyzed in triplicate using TaqMan Fast Universal PCR Master Mix (Life Technologies, 4352042) with the following TaqMan gene specific primer-(FAM/MGB) probe mixes (Life Technologies): PML (assay ID Hs00231241_m1), IFI16 (assay ID Hs00986757_m1), ATRX (assay ID HS00997529_m1), Mx1 (assay ID HS00895608_m1) ISG15 (assay ID HS01921425_s1), ISG54 (assay ID Hs01922738_s1), or GAPDH (4333764F) on a 7500 Fast Real time PCR system (Applied Biosystems). .. Reverse transcription (RT) was performed using the TaqMan Reverse Transcription Reagents kit (Life Technologies, N8080234) with oligo(dT) primers. cDNA samples were analyzed in triplicate using TaqMan Fast Universal PCR Master Mix (Life Technologies, 4352042) with the following TaqMan gene specific primer-(FAM/MGB) probe mixes (Life Technologies): PML (assay ID Hs00231241_m1), IFI16 (assay ID Hs00986757_m1), ATRX (assay ID HS00997529_m1), Mx1 (assay ID HS00895608_m1) ISG15 (assay ID HS01921425_s1), ISG54 (assay ID Hs01922738_s1), or GAPDH (4333764F) on a 7500 Fast Real time PCR system (Applied Biosystems).

    Quantitation Assay:

    Article Title: Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infectionSpatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy
    Article Snippet: Reverse transcription (RT) was performed using the TaqMan Reverse Transcription Reagents kit (Life Technologies, N8080234) with oligo(dT) primers. cDNA samples were analyzed in triplicate using TaqMan Fast Universal PCR Master Mix (Life Technologies, 4352042) with the following TaqMan gene specific primer-(FAM/MGB) probe mixes (Life Technologies): PML (assay ID Hs00231241_m1), IFI16 (assay ID Hs00986757_m1), ATRX (assay ID HS00997529_m1), Mx1 (assay ID HS00895608_m1) ISG15 (assay ID HS01921425_s1), ISG54 (assay ID Hs01922738_s1), or GAPDH (4333764F) on a 7500 Fast Real time PCR system (Applied Biosystems). .. Reverse transcription (RT) was performed using the TaqMan Reverse Transcription Reagents kit (Life Technologies, N8080234) with oligo(dT) primers. cDNA samples were analyzed in triplicate using TaqMan Fast Universal PCR Master Mix (Life Technologies, 4352042) with the following TaqMan gene specific primer-(FAM/MGB) probe mixes (Life Technologies): PML (assay ID Hs00231241_m1), IFI16 (assay ID Hs00986757_m1), ATRX (assay ID HS00997529_m1), Mx1 (assay ID HS00895608_m1) ISG15 (assay ID HS01921425_s1), ISG54 (assay ID Hs01922738_s1), or GAPDH (4333764F) on a 7500 Fast Real time PCR system (Applied Biosystems).

    Polymerase Chain Reaction:

    Article Title: Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infectionSpatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy
    Article Snippet: Total RNA was isolated using the RNAeasy Plus Kit (Qiagen, 74134) according to the manufacturer’s instructions. .. Reverse transcription (RT) was performed using the TaqMan Reverse Transcription Reagents kit (Life Technologies, N8080234) with oligo(dT) primers. cDNA samples were analyzed in triplicate using TaqMan Fast Universal PCR Master Mix (Life Technologies, 4352042) with the following TaqMan gene specific primer-(FAM/MGB) probe mixes (Life Technologies): PML (assay ID Hs00231241_m1), IFI16 (assay ID Hs00986757_m1), ATRX (assay ID HS00997529_m1), Mx1 (assay ID HS00895608_m1) ISG15 (assay ID HS01921425_s1), ISG54 (assay ID Hs01922738_s1), or GAPDH (4333764F) on a 7500 Fast Real time PCR system (Applied Biosystems). .. Relative mRNA levels were determined using the ΔΔCt method, normalized to GAPDH, and expressed relative to indicated treatments.

    Article Title: The effects of substance P and acetylcholine on human tenocyte proliferation converge mechanistically via TGF-β1
    Article Snippet: RNA conversion to cDNA was performed according to the manufacturer’s recommendations: 10 min at 25°C followed by 120 min at 37°C and 5 min at 85°C before idling for 60 min at 4°C in a thermal cycler (MJ Research PTC-200 Thermal Cycler). .. Quantitative PCR was performed using the TaqMan Fast Universal PCR Master Mix (Applied Biosystems, code: 4352042) by splitting the starting volume of 25μl into two 10μl volumes in each level for technical duplicate. .. The levels of TGF- β1 (ABI, code: Hs00998133), TAC1 (ABI, code: Hs00243225), TACR1 (ABI, code: Hs00185530), CHRM1 (ABI, code: Hs00912795), CHMR2 (ABI, code: Hs00265208), CHRM3 (ABI, code: Hs00265216), CHMR4 (ABI, code: Hs00265219), and CHRM5 (ABI, code: Hs00255278) were determined relative to β-actin (ABI, code: 4333762F).

    Article Title: HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth disease
    Article Snippet: For RT-qPCR analysis, three different reference genes and two target genes were selected for evaluation of their expression profile using exon-spanning TaqMan™ gene expression assays (Applied Biosystems): polymerase (RNA) II Subunit A (Polr2a ), hypoxanthine-guanine phosphoribosyltransferase (Hprt ), glyceraldehyde 3-phosphate dehydrogenase (Gapdh ), glycyl-tRNA synthetase (Gars ) and histone deacetylase 6 (Hdac6 ). .. A master mix for each PCR run was prepared containing TaqMan™ Fast Universal PCR Master Mix (2×, no AmpErase UNG, Applied Biosystems) and 1/20 diluted cDNA was added. .. All samples were amplified in triplicate from the same cDNA preparation and the mean values were calculated.

    Article Title: Unusual Five Copies and Dual Forms of nrdB in “Candidatus Liberibacter asiaticus”: Biological Implications and PCR Detection Application
    Article Snippet: The fluorescence signal was captured at the end of each 60 °C step, followed by a melting point analysis. .. For TaqMan® real-time PCR, the reaction mixture contained 10 μl of TaqMan® Fast Universal PCR Master Mix (2X) (Applied Biosystems), 1 μl of DNA template (~25 ng), 0.2 μl of TaqMan® probe (5 μM), 0.4 μl of each forward and reverse primer (10 μM) in a final volume of 20 μl with the following procedure: 50 °C for 2 min, then 95 °C for 20 s, followed by 40 cycles at 95 °C for 3 s and 60 °C for 30 s. The fluorescence signal was captured at the end of each 60 °C step. .. The data were analyzed using Opticon Monitor™ software (MJ Research), StepOnePlus™ Software v2.3 (Applied Biosystems) and Bio-Rad CFX Manager 2.1 software with automated baseline settings and a manually set threshold at 0.1.

    Infection:

    Article Title: Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infectionSpatiotemporal dynamics of HSV genome nuclear entry and compaction state transitions using bioorthogonal chemistry and super-resolution microscopy
    Article Snippet: Reverse transcription (RT) was performed using the TaqMan Reverse Transcription Reagents kit (Life Technologies, N8080234) with oligo(dT) primers. cDNA samples were analyzed in triplicate using TaqMan Fast Universal PCR Master Mix (Life Technologies, 4352042) with the following TaqMan gene specific primer-(FAM/MGB) probe mixes (Life Technologies): PML (assay ID Hs00231241_m1), IFI16 (assay ID Hs00986757_m1), ATRX (assay ID HS00997529_m1), Mx1 (assay ID HS00895608_m1) ISG15 (assay ID HS01921425_s1), ISG54 (assay ID Hs01922738_s1), or GAPDH (4333764F) on a 7500 Fast Real time PCR system (Applied Biosystems). .. Reverse transcription (RT) was performed using the TaqMan Reverse Transcription Reagents kit (Life Technologies, N8080234) with oligo(dT) primers. cDNA samples were analyzed in triplicate using TaqMan Fast Universal PCR Master Mix (Life Technologies, 4352042) with the following TaqMan gene specific primer-(FAM/MGB) probe mixes (Life Technologies): PML (assay ID Hs00231241_m1), IFI16 (assay ID Hs00986757_m1), ATRX (assay ID HS00997529_m1), Mx1 (assay ID HS00895608_m1) ISG15 (assay ID HS01921425_s1), ISG54 (assay ID Hs01922738_s1), or GAPDH (4333764F) on a 7500 Fast Real time PCR system (Applied Biosystems).

    Expressing:

    Article Title: HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth disease
    Article Snippet: For RT-qPCR analysis, three different reference genes and two target genes were selected for evaluation of their expression profile using exon-spanning TaqMan™ gene expression assays (Applied Biosystems): polymerase (RNA) II Subunit A (Polr2a ), hypoxanthine-guanine phosphoribosyltransferase (Hprt ), glyceraldehyde 3-phosphate dehydrogenase (Gapdh ), glycyl-tRNA synthetase (Gars ) and histone deacetylase 6 (Hdac6 ). .. A master mix for each PCR run was prepared containing TaqMan™ Fast Universal PCR Master Mix (2×, no AmpErase UNG, Applied Biosystems) and 1/20 diluted cDNA was added.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: The effects of substance P and acetylcholine on human tenocyte proliferation converge mechanistically via TGF-β1
    Article Snippet: RNA isolation, reverse transcription and RT-PCR of Achilles tendon cell cultures were performed as previously described [ ]. .. Quantitative PCR was performed using the TaqMan Fast Universal PCR Master Mix (Applied Biosystems, code: 4352042) by splitting the starting volume of 25μl into two 10μl volumes in each level for technical duplicate.

    Reverse Transcriptase Assay:

    Article Title: HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth disease
    Article Snippet: Reverse transcription was performed with the SuperScript® III Reverse Transcriptase assay (Thermo Fisher Scientific) according to the manufacturer’s instructions, using 0.5 µg total RNA. .. A master mix for each PCR run was prepared containing TaqMan™ Fast Universal PCR Master Mix (2×, no AmpErase UNG, Applied Biosystems) and 1/20 diluted cDNA was added.

    Software:

    Article Title: HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth disease
    Article Snippet: A master mix for each PCR run was prepared containing TaqMan™ Fast Universal PCR Master Mix (2×, no AmpErase UNG, Applied Biosystems) and 1/20 diluted cDNA was added. .. All samples were amplified in triplicate from the same cDNA preparation and the mean values were calculated.

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    Thermo Fisher taqman fast universal pcr master mix
    30% CR affected the MicroRNA expression in mouse liver The expression of indicated miRNAs was assayed using ( A ) microarray hybridization and ( B ) <t>Taqman</t> <t>PCR</t> assay in the liver of AL (Open bars) and CR mice (Black bars). * - statistically significant difference (p
    Taqman Fast Universal Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 313 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taqman fast universal pcr master mix/product/Thermo Fisher
    Average 99 stars, based on 313 article reviews
    Price from $9.99 to $1999.99
    taqman fast universal pcr master mix - by Bioz Stars, 2020-01
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    87
    Thermo Fisher gene exp dyx1c1 ccpg1 hs00370049 m1
    Knockdown of RFX1 , RFX2 , and RFX3 affect the expression of <t>DYX1C1</t> and DCDC2 but not KIAA0319 in hTERT-RPE1 cells. A ) Binding of RFX1 and RFX2 to the X-box motifs present in DYX1C1 and DCDC2 promoters. Biotinylated probes spanning the X-box motifs were incubated with nuclear extracts from serum-starved hTERT-RPE1 cells with or without antibodies (ab) against RFX1, RFX2, and RFX3. Supershifts for both probes were detected for RFX1 and RFX2 antibodies (white arrowheads). One representative experiment of 3 is shown. B ) Fold-change differences in the expression of DCGs upon knockdown of RFX TFs. By using siRNA against RFX1 , RFX2 , and RFX3 , alone or in different combinations, genes were silenced in hTERT-RPE1 cells. Cells were thereafter starved for 24 h to induce ciliogenesis, and expression levels of DYX1C1 , DCDC2 , and KIAA0319 were measured by using qRT-PCR. C ) Fold-change difference in expression levels of RFX1 , RFX2 , and RFX3 upon siRNA silencing. qRT-PCR data are summarized by using the ΔΔC t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem . Significance of the post hoc tests after ANOVA is presented as *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05 after multiple correction of simultaneous comparisons.
    Gene Exp Dyx1c1 Ccpg1 Hs00370049 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp dyx1c1 ccpg1 hs00370049 m1/product/Thermo Fisher
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    Image Search Results


    30% CR affected the MicroRNA expression in mouse liver The expression of indicated miRNAs was assayed using ( A ) microarray hybridization and ( B ) Taqman PCR assay in the liver of AL (Open bars) and CR mice (Black bars). * - statistically significant difference (p

    Journal: Aging (Albany NY)

    Article Title: Aging and calorie restriction regulate the expression of miR-125a-5p and its target genes Stat3, Casp2 and Stard13

    doi: 10.18632/aging.101270

    Figure Lengend Snippet: 30% CR affected the MicroRNA expression in mouse liver The expression of indicated miRNAs was assayed using ( A ) microarray hybridization and ( B ) Taqman PCR assay in the liver of AL (Open bars) and CR mice (Black bars). * - statistically significant difference (p

    Article Snippet: Determination of miRNA expression of miR-125a-5p and miR-145b was done by using TaqMan micro RNA assay (ThermoFisher Scientific Catalog # 4427975), and TaqMan Fast Universal PCR master mix 2x (ThermoFisher Scientific Catalog # 4352042) as per the manufacturer's instructions.

    Techniques: Expressing, Microarray, Hybridization, Polymerase Chain Reaction, Mouse Assay

    Expression of GR in clinical lung adenocarcinoma and cell line models. A lung adenocarcinoma tissue microarray was probed by immunohistochemistry for expression of GR and the expression levels were scored in the malignant cells by intensity of staining as well as percent of cells that stained positive (Panel A). Representative images showing the different intensities of staining for GR are shown (Panel B). Whole cell lysates were extracted from H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells and GR expression was visualized by western blot using GAPDH as the loading control (Panel C). RNA was also extracted from the same cell lines and the levels of GR mRNA were measured by real time RT-PCR using TaqMan probes and plotted on a linear scale (Panel D). GR mRNA was measured by quantitative RT-PCR using SYBR Green in a cDNA microarray of clinical lung adenocarcinoma tumors; the relative levels of GR mRNA in H1299 and H1299GR Clone 4 cells were simultaneously measured using SYBR Green and the data plotted on a log scale (dashed lines) (Panel E). Panel D: *P, 0.002; **P, 0.001; ***P, 0.003.

    Journal: Scientific Reports

    Article Title: Chronic p27Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

    doi: 10.1038/s41598-018-34475-8

    Figure Lengend Snippet: Expression of GR in clinical lung adenocarcinoma and cell line models. A lung adenocarcinoma tissue microarray was probed by immunohistochemistry for expression of GR and the expression levels were scored in the malignant cells by intensity of staining as well as percent of cells that stained positive (Panel A). Representative images showing the different intensities of staining for GR are shown (Panel B). Whole cell lysates were extracted from H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells and GR expression was visualized by western blot using GAPDH as the loading control (Panel C). RNA was also extracted from the same cell lines and the levels of GR mRNA were measured by real time RT-PCR using TaqMan probes and plotted on a linear scale (Panel D). GR mRNA was measured by quantitative RT-PCR using SYBR Green in a cDNA microarray of clinical lung adenocarcinoma tumors; the relative levels of GR mRNA in H1299 and H1299GR Clone 4 cells were simultaneously measured using SYBR Green and the data plotted on a log scale (dashed lines) (Panel E). Panel D: *P, 0.002; **P, 0.001; ***P, 0.003.

    Article Snippet: Reverse transcription was performed using 500 ng of total RNA and High-Capacity cDNA Archive kit (Thermo Fisher Scientific), according to the vendor’s protocol. cDNA was measured by quantitative real-time PCR using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) and TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific).

    Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Western Blot, Quantitative RT-PCR, SYBR Green Assay

    Knockdown of RFX1 , RFX2 , and RFX3 affect the expression of DYX1C1 and DCDC2 but not KIAA0319 in hTERT-RPE1 cells. A ) Binding of RFX1 and RFX2 to the X-box motifs present in DYX1C1 and DCDC2 promoters. Biotinylated probes spanning the X-box motifs were incubated with nuclear extracts from serum-starved hTERT-RPE1 cells with or without antibodies (ab) against RFX1, RFX2, and RFX3. Supershifts for both probes were detected for RFX1 and RFX2 antibodies (white arrowheads). One representative experiment of 3 is shown. B ) Fold-change differences in the expression of DCGs upon knockdown of RFX TFs. By using siRNA against RFX1 , RFX2 , and RFX3 , alone or in different combinations, genes were silenced in hTERT-RPE1 cells. Cells were thereafter starved for 24 h to induce ciliogenesis, and expression levels of DYX1C1 , DCDC2 , and KIAA0319 were measured by using qRT-PCR. C ) Fold-change difference in expression levels of RFX1 , RFX2 , and RFX3 upon siRNA silencing. qRT-PCR data are summarized by using the ΔΔC t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem . Significance of the post hoc tests after ANOVA is presented as *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05 after multiple correction of simultaneous comparisons.

    Journal: The FASEB Journal

    Article Title: Ciliary dyslexia candidate genes DYX1C1 andDCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

    doi: 10.1096/fj.201500124RR

    Figure Lengend Snippet: Knockdown of RFX1 , RFX2 , and RFX3 affect the expression of DYX1C1 and DCDC2 but not KIAA0319 in hTERT-RPE1 cells. A ) Binding of RFX1 and RFX2 to the X-box motifs present in DYX1C1 and DCDC2 promoters. Biotinylated probes spanning the X-box motifs were incubated with nuclear extracts from serum-starved hTERT-RPE1 cells with or without antibodies (ab) against RFX1, RFX2, and RFX3. Supershifts for both probes were detected for RFX1 and RFX2 antibodies (white arrowheads). One representative experiment of 3 is shown. B ) Fold-change differences in the expression of DCGs upon knockdown of RFX TFs. By using siRNA against RFX1 , RFX2 , and RFX3 , alone or in different combinations, genes were silenced in hTERT-RPE1 cells. Cells were thereafter starved for 24 h to induce ciliogenesis, and expression levels of DYX1C1 , DCDC2 , and KIAA0319 were measured by using qRT-PCR. C ) Fold-change difference in expression levels of RFX1 , RFX2 , and RFX3 upon siRNA silencing. qRT-PCR data are summarized by using the ΔΔC t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem . Significance of the post hoc tests after ANOVA is presented as *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05 after multiple correction of simultaneous comparisons.

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was analyzed with cDNA diluted 1:5 using TaqMan expression assays and TaqMan fast Universal PCR Master Mix [4352042; Thermo Fisher Scientific;DYX1C1 : Hs00370049_m1; DCDC2 : Hs00393203_m1;KIAA0319 : Hs00207788_m1; HPRT 1 (hypoxanthine phosphoribosyltransferase 1): Hs02800695_m1; CDK1 (cyclin-dependent kinase 1): Hs00938777_m1; TUBA1A (tubulin α1a): Hs00362387_m1; RFX2 : 01100925_m1] or using SYBR green primers for RFX1 , RFX3 , and HPRT1 (10 µM) and the Fast SYBR Green Master Mix (4385612; Applied Biosystems, Foster City, CA, USA).

    Techniques: Expressing, Binding Assay, Incubation, Quantitative RT-PCR

    The promoters of DYX1C1 , DCDC2 , and KIAA0319 contain functional X-box promoter motifs. A ) Location of putative X-box promoter motifs upstream of DYX1C1 , DCDC2 , and KIAA0319 identified in the bioinformatics screen. X-box sequence logos on the basis of human, mouse, dog, cow, and cat sequence alignments are shown above 3 well-conserved motifs. B ) Luciferase expression assays: pGL3 basic vector containing promoter sequences of DYX1C1 , DCDC2 , and KIAA0319 (containing WT or mutated X-box motifs) were transfected into starved hTERT-RPE1 and SH-SY5Y cells under normal growth conditions. Luciferase expression was measured and data are shown as relative ratio to the WT construct. Data are displayed as means ± sem. * P

    Journal: The FASEB Journal

    Article Title: Ciliary dyslexia candidate genes DYX1C1 andDCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

    doi: 10.1096/fj.201500124RR

    Figure Lengend Snippet: The promoters of DYX1C1 , DCDC2 , and KIAA0319 contain functional X-box promoter motifs. A ) Location of putative X-box promoter motifs upstream of DYX1C1 , DCDC2 , and KIAA0319 identified in the bioinformatics screen. X-box sequence logos on the basis of human, mouse, dog, cow, and cat sequence alignments are shown above 3 well-conserved motifs. B ) Luciferase expression assays: pGL3 basic vector containing promoter sequences of DYX1C1 , DCDC2 , and KIAA0319 (containing WT or mutated X-box motifs) were transfected into starved hTERT-RPE1 and SH-SY5Y cells under normal growth conditions. Luciferase expression was measured and data are shown as relative ratio to the WT construct. Data are displayed as means ± sem. * P

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was analyzed with cDNA diluted 1:5 using TaqMan expression assays and TaqMan fast Universal PCR Master Mix [4352042; Thermo Fisher Scientific;DYX1C1 : Hs00370049_m1; DCDC2 : Hs00393203_m1;KIAA0319 : Hs00207788_m1; HPRT 1 (hypoxanthine phosphoribosyltransferase 1): Hs02800695_m1; CDK1 (cyclin-dependent kinase 1): Hs00938777_m1; TUBA1A (tubulin α1a): Hs00362387_m1; RFX2 : 01100925_m1] or using SYBR green primers for RFX1 , RFX3 , and HPRT1 (10 µM) and the Fast SYBR Green Master Mix (4385612; Applied Biosystems, Foster City, CA, USA).

    Techniques: Functional Assay, Sequencing, Luciferase, Expressing, Plasmid Preparation, Transfection, Construct

    Induction of ciliogenesis in hTERT-RPE1 cells affects the expression levels of DCGs, RFX TF genes, and ciliogenesis/cell-cycle marker genes. hTERT-RPE1 cells were starved for 6, 12, and 24 h to induce ciliogenesis. Fold-change of difference for each time point compared with 0 h is shown for DYX1C1 ( A ), DCDC2 ( B ), KIAA0319 ( C ), RFX1 ( D ), RFX2 ( E ), RFX3 ( F ), TUBA1A (tubulin α1a) ( G ), IFT57 (intraflagellar transport 57) ( H ), and CDK1 (cyclin-dependent kinase 1) ( I ). qRT-PCR data are summarized by using the ΔΔ C t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem .

    Journal: The FASEB Journal

    Article Title: Ciliary dyslexia candidate genes DYX1C1 andDCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

    doi: 10.1096/fj.201500124RR

    Figure Lengend Snippet: Induction of ciliogenesis in hTERT-RPE1 cells affects the expression levels of DCGs, RFX TF genes, and ciliogenesis/cell-cycle marker genes. hTERT-RPE1 cells were starved for 6, 12, and 24 h to induce ciliogenesis. Fold-change of difference for each time point compared with 0 h is shown for DYX1C1 ( A ), DCDC2 ( B ), KIAA0319 ( C ), RFX1 ( D ), RFX2 ( E ), RFX3 ( F ), TUBA1A (tubulin α1a) ( G ), IFT57 (intraflagellar transport 57) ( H ), and CDK1 (cyclin-dependent kinase 1) ( I ). qRT-PCR data are summarized by using the ΔΔ C t method as displayed as mean fold-change (2 −ΔΔ Ct or −2 ΔΔ Ct ) ± sem .

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was analyzed with cDNA diluted 1:5 using TaqMan expression assays and TaqMan fast Universal PCR Master Mix [4352042; Thermo Fisher Scientific;DYX1C1 : Hs00370049_m1; DCDC2 : Hs00393203_m1;KIAA0319 : Hs00207788_m1; HPRT 1 (hypoxanthine phosphoribosyltransferase 1): Hs02800695_m1; CDK1 (cyclin-dependent kinase 1): Hs00938777_m1; TUBA1A (tubulin α1a): Hs00362387_m1; RFX2 : 01100925_m1] or using SYBR green primers for RFX1 , RFX3 , and HPRT1 (10 µM) and the Fast SYBR Green Master Mix (4385612; Applied Biosystems, Foster City, CA, USA).

    Techniques: Expressing, Marker, Quantitative RT-PCR

    Subcellular localization of endogenous DYX1C1 ( A ) and DCDC2 ( B ) in hTERT-RPE1 cells. hTERT-RPE1 cells were grown to 80% confluence, serum starved for 24 h to induce ciliogenesis, then fixed and stained for DYX1C1, DCDC2, acetylated α-tubulin (cilia marker), or γ-tubulin (centrosome marker). Nuclei are stained with DRAQ5. Scale bars, 20 µm, 2 µm (insets).

    Journal: The FASEB Journal

    Article Title: Ciliary dyslexia candidate genes DYX1C1 andDCDC2 are regulated by Regulatory Factor X (RFX) transcription factors through X-box promoter motifs

    doi: 10.1096/fj.201500124RR

    Figure Lengend Snippet: Subcellular localization of endogenous DYX1C1 ( A ) and DCDC2 ( B ) in hTERT-RPE1 cells. hTERT-RPE1 cells were grown to 80% confluence, serum starved for 24 h to induce ciliogenesis, then fixed and stained for DYX1C1, DCDC2, acetylated α-tubulin (cilia marker), or γ-tubulin (centrosome marker). Nuclei are stained with DRAQ5. Scale bars, 20 µm, 2 µm (insets).

    Article Snippet: Quantitative real-time PCR (qRT-PCR) was analyzed with cDNA diluted 1:5 using TaqMan expression assays and TaqMan fast Universal PCR Master Mix [4352042; Thermo Fisher Scientific;DYX1C1 : Hs00370049_m1; DCDC2 : Hs00393203_m1;KIAA0319 : Hs00207788_m1; HPRT 1 (hypoxanthine phosphoribosyltransferase 1): Hs02800695_m1; CDK1 (cyclin-dependent kinase 1): Hs00938777_m1; TUBA1A (tubulin α1a): Hs00362387_m1; RFX2 : 01100925_m1] or using SYBR green primers for RFX1 , RFX3 , and HPRT1 (10 µM) and the Fast SYBR Green Master Mix (4385612; Applied Biosystems, Foster City, CA, USA).

    Techniques: Staining, Marker