Structured Review

Roche long range pcr
Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective <t>HLA</t> gene haplotype phasing. The size selection also determines an actual molar concentration for bridge <t>PCR</t> to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.
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1) Product Images from "Phase-defined complete sequencing of the HLA genes by next-generation sequencing"

Article Title: Phase-defined complete sequencing of the HLA genes by next-generation sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-355

Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.
Figure Legend Snippet: Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.

Techniques Used: Selection, Agarose Gel Electrophoresis, Sample Prep, Concentration Assay, Bridge PCR, Amplification

2) Product Images from "High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic"

Article Title: High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic

Journal: BMC Medical Genetics

doi: 10.1186/1471-2350-8-32

Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 1A/1B-2 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exons 5–14 by long-range PCR and sequencing of the breakpoints. (C) Confirmation of the deletion of exons 11–12 by long-range PCR and sequencing of the breakpoints. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).
Figure Legend Snippet: Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 1A/1B-2 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exons 5–14 by long-range PCR and sequencing of the breakpoints. (C) Confirmation of the deletion of exons 11–12 by long-range PCR and sequencing of the breakpoints. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

Techniques Used: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 18–19 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exon 20 and sequencing of the breakpoints.Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).
Figure Legend Snippet: Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 18–19 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exon 20 and sequencing of the breakpoints.Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

Techniques Used: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

Confirmation and characterization of the rearrangements . Confirmation of the deletion of the exons 21–22 by long-range PCR and sequencing of the breakpoints. The deletion/insertion event was characterized as g.77128_80906del3779ins236. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).
Figure Legend Snippet: Confirmation and characterization of the rearrangements . Confirmation of the deletion of the exons 21–22 by long-range PCR and sequencing of the breakpoints. The deletion/insertion event was characterized as g.77128_80906del3779ins236. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

Techniques Used: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

3) Product Images from "Phase-defined complete sequencing of the HLA genes by next-generation sequencing"

Article Title: Phase-defined complete sequencing of the HLA genes by next-generation sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-355

Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.
Figure Legend Snippet: Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.

Techniques Used: Selection, Agarose Gel Electrophoresis, Sample Prep, Concentration Assay, Bridge PCR, Amplification

4) Product Images from "Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression"

Article Title: Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.18.5611-5626.2003

PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.
Figure Legend Snippet: PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.

Techniques Used: Polymerase Chain Reaction, Amplification

5) Product Images from "A Gata4 nuclear GFP transcriptional reporter to study endoderm and cardiac development in the mouse"

Article Title: A Gata4 nuclear GFP transcriptional reporter to study endoderm and cardiac development in the mouse

Journal: Biology Open

doi: 10.1242/bio.036517

Generation of Gata4 H2B-GFP/+ targeted mouse model. (A) The cassette LoxP-H2B-GFP-4XPloyA-LoxP-FRT-Neo-FRT-3XFLAG was inserted into the start codon of the mouse Gata4 locus through homologous recombination. The Neo cassette was flanked by two FRT sites and removed by crossing to Flippase mice to generate the Gata4 H2B-GFP allele. Cre excision of H2B-GFP generates GATA4-FLAG N-terminal protein fusion Gata4 FLAG allele. (B) The targeted ES cells were confirmed by long-range PCR amplification of two fragments using primers P1 and P2, P3 and P4, respectively. Primers P1 and P2 produced a 4.3 kb band. Primers P3 and P4 produced a 4.9 kb band. (C) Western blot of individual E7.5 embryo littermates from a Gata4 FLAG/+ ×wild-type mating. The anti-FLAG antibody cross reacts with GATA4-FLAG in Gata4 FLAG/+ embryos. Total protein lysis of an ear from a Gata4 FLAG/+ adult male was used as a positive control.
Figure Legend Snippet: Generation of Gata4 H2B-GFP/+ targeted mouse model. (A) The cassette LoxP-H2B-GFP-4XPloyA-LoxP-FRT-Neo-FRT-3XFLAG was inserted into the start codon of the mouse Gata4 locus through homologous recombination. The Neo cassette was flanked by two FRT sites and removed by crossing to Flippase mice to generate the Gata4 H2B-GFP allele. Cre excision of H2B-GFP generates GATA4-FLAG N-terminal protein fusion Gata4 FLAG allele. (B) The targeted ES cells were confirmed by long-range PCR amplification of two fragments using primers P1 and P2, P3 and P4, respectively. Primers P1 and P2 produced a 4.3 kb band. Primers P3 and P4 produced a 4.9 kb band. (C) Western blot of individual E7.5 embryo littermates from a Gata4 FLAG/+ ×wild-type mating. The anti-FLAG antibody cross reacts with GATA4-FLAG in Gata4 FLAG/+ embryos. Total protein lysis of an ear from a Gata4 FLAG/+ adult male was used as a positive control.

Techniques Used: Homologous Recombination, Mouse Assay, Polymerase Chain Reaction, Amplification, Produced, Western Blot, Lysis, Positive Control

6) Product Images from "Phase-defined complete sequencing of the HLA genes by next-generation sequencing"

Article Title: Phase-defined complete sequencing of the HLA genes by next-generation sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-355

Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.
Figure Legend Snippet: Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.

Techniques Used: Selection, Agarose Gel Electrophoresis, Sample Prep, Concentration Assay, Bridge PCR, Amplification

7) Product Images from "Mitochondrial DNA deletions and depletion within paraspinal muscles"

Article Title: Mitochondrial DNA deletions and depletion within paraspinal muscles

Journal: Neuropathology and Applied Neurobiology

doi: 10.1111/j.1365-2990.2012.01290.x

Confirmation of mitochondrial DNA (mtDNA) deletions and identification of breakpoints. ( A – B ) When DNA extracted from individual respiratory-deficient paraspinal muscle fibres was subjected to long-range polymerase chain reaction (PCR), multiple mtDNA deletions were detected. However, more than one or two mtDNA deletions within an individual fibre was rarely identified, suggestive of clonally expanded mtDNA deletions ( A ). When DNA was extracted from 10 pooled fibres with intact mitochondrial respiratory chain complex IV or cytochrome c oxidase (COX) activity, multiple mtDNA deletions were also detected ( B ); however, real-time data shown in Figure 2 did not indicate expansion of mtDNA deletions to high heteroplasmy levels in such fibres. COX-deficient: fibres lacking complex IV and with intact complex II activity. COX-positive: fibres with complex IV activity. + indicates full-length amplified product from wild-type DNA. Lanes 1–8 ( A ) show amplified products of DNA extracted from individual fibres from different cases and lanes 1–6 ( B ) show 10 pooled fibres, from different cases. ( C ) Sequencing of individual mtDNA deletions extracted following long-range PCR confirmed the mtDNA deletions and identified breakpoints. MtDNA deletions were flanked by either a perfect ( Ci ) or an imperfect ( Cii ) repeat sequence as previously reported in ageing [ 23 ].
Figure Legend Snippet: Confirmation of mitochondrial DNA (mtDNA) deletions and identification of breakpoints. ( A – B ) When DNA extracted from individual respiratory-deficient paraspinal muscle fibres was subjected to long-range polymerase chain reaction (PCR), multiple mtDNA deletions were detected. However, more than one or two mtDNA deletions within an individual fibre was rarely identified, suggestive of clonally expanded mtDNA deletions ( A ). When DNA was extracted from 10 pooled fibres with intact mitochondrial respiratory chain complex IV or cytochrome c oxidase (COX) activity, multiple mtDNA deletions were also detected ( B ); however, real-time data shown in Figure 2 did not indicate expansion of mtDNA deletions to high heteroplasmy levels in such fibres. COX-deficient: fibres lacking complex IV and with intact complex II activity. COX-positive: fibres with complex IV activity. + indicates full-length amplified product from wild-type DNA. Lanes 1–8 ( A ) show amplified products of DNA extracted from individual fibres from different cases and lanes 1–6 ( B ) show 10 pooled fibres, from different cases. ( C ) Sequencing of individual mtDNA deletions extracted following long-range PCR confirmed the mtDNA deletions and identified breakpoints. MtDNA deletions were flanked by either a perfect ( Ci ) or an imperfect ( Cii ) repeat sequence as previously reported in ageing [ 23 ].

Techniques Used: Polymerase Chain Reaction, Activity Assay, Amplification, Sequencing

Total and wild-type mtDNA copies in individual paraspinal muscle fibres. ( A ) Ratio of total mitochondrial DNA (mtDNA) copies in respiratory-deficient paraspinal muscle fibres [wild-type and deleted based on MTND1 values in real-time polymerase chain reaction (PCR)], corrected for case to case variability by using the mean copy number of 10 fibres with intact mitochondrial respiratory chain complex IV or cytochrome c oxidase (COX) activity (COX-positive) in each case, indicated a significantly greater total mtDNA copy number compared with COX-positive fibres ( A ). When the lower limit of 95% confidence interval of total copy number ratios in COX-positive fibres were used to identify fibres with mtDNA depletion ( A , below the dotted line), we detected a number of respiratory-deficient fibres (13.5% of all respiratory-deficient fibres) where total mtDNA copies were depleted in the absence of high level of mtDNA deletion. ( B ) Ratio of wild-type mtDNA copies in respiratory-deficient paraspinal muscle fibres (based on MTND4 values in real-time PCR), corrected for variability in wild-type mtDNA copies within fibres with intact COX activity (COX-positive), indicated a significantly decrease in wild-type mtDNA copies in respiratory-deficient fibres with high heteroplasmy levels compared with COX-positive fibres ( B ). The increase in total mtDNA copies and decrease in wild-type mtDNA copies in respiratory-deficient fibres were consistent with pathogenecity of high levels of mtDNA deletion, previously reported [ 12 , 19 ]. When the lower limit of 95% confidence interval of wild type copy number ratios in COX-positive fibres were used to identify fibres with mtDNA depletion ( B , below the dotted line), a number of respiratory-deficient fibres (11.4% of all respiratory-deficient fibres) with depletion of wild type mtDNA copies and without high level of mtDNA deletion was detected.
Figure Legend Snippet: Total and wild-type mtDNA copies in individual paraspinal muscle fibres. ( A ) Ratio of total mitochondrial DNA (mtDNA) copies in respiratory-deficient paraspinal muscle fibres [wild-type and deleted based on MTND1 values in real-time polymerase chain reaction (PCR)], corrected for case to case variability by using the mean copy number of 10 fibres with intact mitochondrial respiratory chain complex IV or cytochrome c oxidase (COX) activity (COX-positive) in each case, indicated a significantly greater total mtDNA copy number compared with COX-positive fibres ( A ). When the lower limit of 95% confidence interval of total copy number ratios in COX-positive fibres were used to identify fibres with mtDNA depletion ( A , below the dotted line), we detected a number of respiratory-deficient fibres (13.5% of all respiratory-deficient fibres) where total mtDNA copies were depleted in the absence of high level of mtDNA deletion. ( B ) Ratio of wild-type mtDNA copies in respiratory-deficient paraspinal muscle fibres (based on MTND4 values in real-time PCR), corrected for variability in wild-type mtDNA copies within fibres with intact COX activity (COX-positive), indicated a significantly decrease in wild-type mtDNA copies in respiratory-deficient fibres with high heteroplasmy levels compared with COX-positive fibres ( B ). The increase in total mtDNA copies and decrease in wild-type mtDNA copies in respiratory-deficient fibres were consistent with pathogenecity of high levels of mtDNA deletion, previously reported [ 12 , 19 ]. When the lower limit of 95% confidence interval of wild type copy number ratios in COX-positive fibres were used to identify fibres with mtDNA depletion ( B , below the dotted line), a number of respiratory-deficient fibres (11.4% of all respiratory-deficient fibres) with depletion of wild type mtDNA copies and without high level of mtDNA deletion was detected.

Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Activity Assay

8) Product Images from "Extensive Variation in Gene Copy Number at the Killer Immunoglobulin-Like Receptor Locus in Humans"

Article Title: Extensive Variation in Gene Copy Number at the Killer Immunoglobulin-Like Receptor Locus in Humans

Journal: PLoS ONE

doi: 10.1371/journal.pone.0067619

Validation of the accuracy of genotyping with MLPA by next-generation sequencing (NGS). Five of the genomes with mismatches between MLPA and PCR-SSP were analyzed by NGS. The percentage of reads that aligned against a reference for a KIR gene gives an impression about the presence of the KIR gene in that specific donor. Circles indicate the mismatches found between MLPA and PCR-SSP. KIR3DL3 is a control gene as it is present in all donors.
Figure Legend Snippet: Validation of the accuracy of genotyping with MLPA by next-generation sequencing (NGS). Five of the genomes with mismatches between MLPA and PCR-SSP were analyzed by NGS. The percentage of reads that aligned against a reference for a KIR gene gives an impression about the presence of the KIR gene in that specific donor. Circles indicate the mismatches found between MLPA and PCR-SSP. KIR3DL3 is a control gene as it is present in all donors.

Techniques Used: Multiplex Ligation-dependent Probe Amplification, Next-Generation Sequencing, Polymerase Chain Reaction

9) Product Images from "Misbehaviour of XIST RNA in Breast Cancer Cells"

Article Title: Misbehaviour of XIST RNA in Breast Cancer Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0005559

Effects of BRCA1 RNAi on XIST expression in cells with different XCI status. HMEC (XCI type 0), MCF7 (XCI type 1) and T47D (XCI type 2) were transfected with a mix of two BRCA1 -specific siRNAs, mapping to exons 12 and 24, or a control siRNA. After 72 hrs, cells were processed for BRCA1 immunofluorescence and RNA purification. In all panels BRCA1 is immunostained in green and nuclei are marked with DAPI. The histogram represents quantitative RT-PCR analysis performed on cDNAs of the indicated cell lines, before and after BRCA1 silencing, using primers specific for spliced and unspliced XIST RNA. XIST RNA levels are expressed as a ratio to GAPDH mRNA levels, after subtraction of the background signal from cDNA synthesis reactions lacking reverse transcriptase. To facilitate comparison between cell lines with different XCI status, the XIST / GAPDH transcript ratio was normalised relative to HMEC. Error bars represent standard deviation and the asterisks indicate statistically significant differences (p
Figure Legend Snippet: Effects of BRCA1 RNAi on XIST expression in cells with different XCI status. HMEC (XCI type 0), MCF7 (XCI type 1) and T47D (XCI type 2) were transfected with a mix of two BRCA1 -specific siRNAs, mapping to exons 12 and 24, or a control siRNA. After 72 hrs, cells were processed for BRCA1 immunofluorescence and RNA purification. In all panels BRCA1 is immunostained in green and nuclei are marked with DAPI. The histogram represents quantitative RT-PCR analysis performed on cDNAs of the indicated cell lines, before and after BRCA1 silencing, using primers specific for spliced and unspliced XIST RNA. XIST RNA levels are expressed as a ratio to GAPDH mRNA levels, after subtraction of the background signal from cDNA synthesis reactions lacking reverse transcriptase. To facilitate comparison between cell lines with different XCI status, the XIST / GAPDH transcript ratio was normalised relative to HMEC. Error bars represent standard deviation and the asterisks indicate statistically significant differences (p

Techniques Used: Expressing, Transfection, Immunofluorescence, Purification, Quantitative RT-PCR, Standard Deviation

XIST expression and status of X chromosomes and BRCA1 in HMEC and breast cancer cell lines, and evaluation of XIST levels in different groups of breast carcinomas. A) Classification of HMEC and breast cancer cell lines according to XCI type, based on the indicated X chromosome related features. BRCA1 status is also reported. B) Box-plots of the log2-transformed amounts of XIST RNA measured by quantitative real-time RT-PCR in the indicated groups of primary human breast cancers. Each box-plot represents the first quartile (lower edge of the box), median value (bar inside the box), third quartile (upper edge of the box), and minimum and maximum values (horizontal lines). Points at a distance from the quartiles > 1.5 times the inter-quartile range are plotted individually. Statistically significant p values between groups are reported (Kruskal-Wallis Rank Sum test).
Figure Legend Snippet: XIST expression and status of X chromosomes and BRCA1 in HMEC and breast cancer cell lines, and evaluation of XIST levels in different groups of breast carcinomas. A) Classification of HMEC and breast cancer cell lines according to XCI type, based on the indicated X chromosome related features. BRCA1 status is also reported. B) Box-plots of the log2-transformed amounts of XIST RNA measured by quantitative real-time RT-PCR in the indicated groups of primary human breast cancers. Each box-plot represents the first quartile (lower edge of the box), median value (bar inside the box), third quartile (upper edge of the box), and minimum and maximum values (horizontal lines). Points at a distance from the quartiles > 1.5 times the inter-quartile range are plotted individually. Statistically significant p values between groups are reported (Kruskal-Wallis Rank Sum test).

Techniques Used: Expressing, Transformation Assay, Quantitative RT-PCR

10) Product Images from "Widespread somatic L1 retrotransposition occurs early during gastrointestinal cancer evolution"

Article Title: Widespread somatic L1 retrotransposition occurs early during gastrointestinal cancer evolution

Journal: Genome Research

doi: 10.1101/gr.196238.115

Representative examples of further somatic L1 insertions in colon cancer patient 2BV and gastric cancer patient 2043. ( A ) L1 insertions in colorectal cancer (all detectable by conventional PCR), from left to right : ins. D2 (polyp-specific, present exclusively
Figure Legend Snippet: Representative examples of further somatic L1 insertions in colon cancer patient 2BV and gastric cancer patient 2043. ( A ) L1 insertions in colorectal cancer (all detectable by conventional PCR), from left to right : ins. D2 (polyp-specific, present exclusively

Techniques Used: Polymerase Chain Reaction

PCR and Sanger-sequencing validation scheme of L1-seq results. ( A ) Multistep PCR validation scheme and location of primers used. Insertions were primarily validated with conventional PCR at their 3′ junction using the L1Hs with the 3′
Figure Legend Snippet: PCR and Sanger-sequencing validation scheme of L1-seq results. ( A ) Multistep PCR validation scheme and location of primers used. Insertions were primarily validated with conventional PCR at their 3′ junction using the L1Hs with the 3′

Techniques Used: Polymerase Chain Reaction, Sequencing

Distribution of somatic L1 insertions. ( A ) Normal colon crypt containing a few cells with L1 insertions (detectable only by nested PCR). ( B ) Colon tumor with early L1 insertions (detectable by conventional PCR). ( C ) L1 insertions occurring late during
Figure Legend Snippet: Distribution of somatic L1 insertions. ( A ) Normal colon crypt containing a few cells with L1 insertions (detectable only by nested PCR). ( B ) Colon tumor with early L1 insertions (detectable by conventional PCR). ( C ) L1 insertions occurring late during

Techniques Used: Nested PCR, Polymerase Chain Reaction

11) Product Images from "C11orf95-MKL2 is the Resulting Fusion Oncogene of t(11;16)(q13;p13) in Chondroid Lipoma"

Article Title: C11orf95-MKL2 is the Resulting Fusion Oncogene of t(11;16)(q13;p13) in Chondroid Lipoma

Journal: Genes, chromosomes & cancer

doi: 10.1002/gcc.20788

A: Schematic of C11orf95 , MKL2 and C11orf95-MKL2 fusion gene, solid bars represent coding exons, gray boxes are non-translated regions. B: Amino acid sequence at the breakpoint. C: Schematic and domain structure of the fusion C11orf95-MKL2 protein; the letters within the bars designate conserved domains; polyglutamic acid region (E), proline rich region (P), DNA-binding SAP domain (SAP), and coiled-coiled region (CC). D: Detection of the presence of C11orf95-MKL2 fusion transcript by RT-PCR analysis.
Figure Legend Snippet: A: Schematic of C11orf95 , MKL2 and C11orf95-MKL2 fusion gene, solid bars represent coding exons, gray boxes are non-translated regions. B: Amino acid sequence at the breakpoint. C: Schematic and domain structure of the fusion C11orf95-MKL2 protein; the letters within the bars designate conserved domains; polyglutamic acid region (E), proline rich region (P), DNA-binding SAP domain (SAP), and coiled-coiled region (CC). D: Detection of the presence of C11orf95-MKL2 fusion transcript by RT-PCR analysis.

Techniques Used: Sequencing, Binding Assay, Reverse Transcription Polymerase Chain Reaction

12) Product Images from "APE1 deficiency promotes cellular senescence and premature aging features"

Article Title: APE1 deficiency promotes cellular senescence and premature aging features

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky326

APE1 knock-down results in elevated genomic DNA damage and increased γ-H2AX foci at telomeres. DNA damage was quantified in the general genome ( A ) or telomeric region ( B ) of APE1-deficient (shAPE1) BJ fibroblasts relative to the scramble control using a targeted PCR strategy. See Figure 2 for general overview of experimental approach. ( C ) Telomere length was assayed by Southern blotting in scramble control (Scr) or APE1 knock-down (shAPE1) BJ fibroblasts. A representative image is shown, and the molecular standards are indicated. ( D ) Expression of γ-H2AX, pATM, 53BP1 and pDNA-PKcs was determined by western blotting in APE1-deficient (shAPE1) and scramble control (Scr) primary fibroblasts (designated). β-actin served as the loading control, and relative expression level (in comparison to relevant scramble control) is designated below each lane. ( E ) Immunofluorescence-FISH of γ-H2AX and PNA probe (Q-FISH) in APE1-deficient (shAPE1) and control (scramble) BJ fibroblasts. Nuclei are designated by yellow dashed line and white arrows indicate colocalization of the two signals. ( F ) Quantitation of the percentage of cells that exhibit > 5 γ-H2AX foci/cell. ( G ) Quantitation of the percentage of overlap between γ-H2AX and telomeric DNA (Q-FISH foci). Graphical results represent averages and standard deviations of three biological replicates with at least four technical replicates each (panel A and B) or three biological replicates (panel F and G). * P -value
Figure Legend Snippet: APE1 knock-down results in elevated genomic DNA damage and increased γ-H2AX foci at telomeres. DNA damage was quantified in the general genome ( A ) or telomeric region ( B ) of APE1-deficient (shAPE1) BJ fibroblasts relative to the scramble control using a targeted PCR strategy. See Figure 2 for general overview of experimental approach. ( C ) Telomere length was assayed by Southern blotting in scramble control (Scr) or APE1 knock-down (shAPE1) BJ fibroblasts. A representative image is shown, and the molecular standards are indicated. ( D ) Expression of γ-H2AX, pATM, 53BP1 and pDNA-PKcs was determined by western blotting in APE1-deficient (shAPE1) and scramble control (Scr) primary fibroblasts (designated). β-actin served as the loading control, and relative expression level (in comparison to relevant scramble control) is designated below each lane. ( E ) Immunofluorescence-FISH of γ-H2AX and PNA probe (Q-FISH) in APE1-deficient (shAPE1) and control (scramble) BJ fibroblasts. Nuclei are designated by yellow dashed line and white arrows indicate colocalization of the two signals. ( F ) Quantitation of the percentage of cells that exhibit > 5 γ-H2AX foci/cell. ( G ) Quantitation of the percentage of overlap between γ-H2AX and telomeric DNA (Q-FISH foci). Graphical results represent averages and standard deviations of three biological replicates with at least four technical replicates each (panel A and B) or three biological replicates (panel F and G). * P -value

Techniques Used: Polymerase Chain Reaction, Southern Blot, Expressing, Western Blot, Immunofluorescence, Fluorescence In Situ Hybridization, Quantitation Assay

13) Product Images from "Extensive somatic L1 retrotransposition in colorectal tumors"

Article Title: Extensive somatic L1 retrotransposition in colorectal tumors

Journal: Genome Research

doi: 10.1101/gr.145235.112

Genomic distribution of L1 insertions. Outer rings show the density of detected insertion sites for reference (gray) and nonreference (black) L1s. The approximate locations of the 72 PCR-validated somatic insertions are indicated by dots inside the circle.
Figure Legend Snippet: Genomic distribution of L1 insertions. Outer rings show the density of detected insertion sites for reference (gray) and nonreference (black) L1s. The approximate locations of the 72 PCR-validated somatic insertions are indicated by dots inside the circle.

Techniques Used: Polymerase Chain Reaction

Analysis of factors influencing L1 activity. ( A ) L1 CpG promoter methylation status performed by quantitative bisulfite PCR analysis. (N) Normal tissue; (T) tumor tissue; (*) MSI. Replicates of four were done for each data point. (Error bars) Standard
Figure Legend Snippet: Analysis of factors influencing L1 activity. ( A ) L1 CpG promoter methylation status performed by quantitative bisulfite PCR analysis. (N) Normal tissue; (T) tumor tissue; (*) MSI. Replicates of four were done for each data point. (Error bars) Standard

Techniques Used: Activity Assay, Methylation, Polymerase Chain Reaction

PCR validation scheme of L1-seq results. ( A ) The three-step PCR validation scheme and location of primers used. Triangles symbolize TSD. ( B ) PCR validation of the 3′ junction (ins. 7). This insertion is in tumor 1 of the eight DNA samples that
Figure Legend Snippet: PCR validation scheme of L1-seq results. ( A ) The three-step PCR validation scheme and location of primers used. Triangles symbolize TSD. ( B ) PCR validation of the 3′ junction (ins. 7). This insertion is in tumor 1 of the eight DNA samples that

Techniques Used: Polymerase Chain Reaction

14) Product Images from "Molecular Genetic Analysis of ICEF, an Integrative Conjugal Element That Is Present as a Repetitive Sequence in the Chromosome of Mycoplasma fermentans PG18"

Article Title: Molecular Genetic Analysis of ICEF, an Integrative Conjugal Element That Is Present as a Repetitive Sequence in the Chromosome of Mycoplasma fermentans PG18

Journal: Journal of Bacteriology

doi: 10.1128/JB.184.24.6929-6941.2002

Detection of an extrachromosomal form of ICEF. (A) Model for integration-excision of ICEF units. The direct repeats flanking ICEF are shown as open rectangles abutting the left (L) and right (R) termini of the integrated form of ICEF (solid bar). In the extrachromosomal form, the termini are juxtaposed and separated by a 6-bp coupling sequence (CS). Orientations of primers (triangles) used to amplify the unique configuration of the extrachromosomal form are indicated. The solid triangles represent primers 7 and 9 (see Materials and Methods) that together amplify terminal regions of extrachromosomal forms of all known ICEF units. PCR with primer 16 (open triangle) in combination with primer 7 (right-facing solid triangle in integrated form) selectively amplified a product from an extrachromosomal form of ICEF-IA. (B) Amplicons derived from genomic DNA containing extrachromosomal ICEF following agarose gel electrophoresis. Lanes: 1, PCR amplicon obtained with primers 7 and 9 with M. fermentans PG18 DNA as the template; 3 to 6, amplicons generated with primers 7 and 16 by using as the template three M. fermentans PG18 DNA preparations purified from different cultures (lanes 3 to 5) or DNA from M. fermentans II-29/I, which lacks ICEF units (lane 6); 2, 1-kb ladder (Promega Corporation, Madison, Wis.). The positions of the anticipated ∼570-bp (primers 7 and 9) and ∼330-bp (primers 7 and 16) amplicons are indicated by arrows at left and right, respectively.
Figure Legend Snippet: Detection of an extrachromosomal form of ICEF. (A) Model for integration-excision of ICEF units. The direct repeats flanking ICEF are shown as open rectangles abutting the left (L) and right (R) termini of the integrated form of ICEF (solid bar). In the extrachromosomal form, the termini are juxtaposed and separated by a 6-bp coupling sequence (CS). Orientations of primers (triangles) used to amplify the unique configuration of the extrachromosomal form are indicated. The solid triangles represent primers 7 and 9 (see Materials and Methods) that together amplify terminal regions of extrachromosomal forms of all known ICEF units. PCR with primer 16 (open triangle) in combination with primer 7 (right-facing solid triangle in integrated form) selectively amplified a product from an extrachromosomal form of ICEF-IA. (B) Amplicons derived from genomic DNA containing extrachromosomal ICEF following agarose gel electrophoresis. Lanes: 1, PCR amplicon obtained with primers 7 and 9 with M. fermentans PG18 DNA as the template; 3 to 6, amplicons generated with primers 7 and 16 by using as the template three M. fermentans PG18 DNA preparations purified from different cultures (lanes 3 to 5) or DNA from M. fermentans II-29/I, which lacks ICEF units (lane 6); 2, 1-kb ladder (Promega Corporation, Madison, Wis.). The positions of the anticipated ∼570-bp (primers 7 and 9) and ∼330-bp (primers 7 and 16) amplicons are indicated by arrows at left and right, respectively.

Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, IA, Derivative Assay, Agarose Gel Electrophoresis, Generated, Purification

Genomic locations of ICEF insertion sites in M. fermentans . The locations of ICEF insertion sites (solid arrows) in M. fermentans PG18 are shown, together with the equivalent region (for ICEF-IIA) of the strain II-29/1 chromosome. The locations and directions of ORFs (open arrows) and individual copies of multicopy ISs ISMi 1 and ISMf 1 (open rectangles) that flank each of the four ICEF units in M. fermentans PG18 are shown. ORFs for coding sequences that either lack homology to known proteins or encode homologs of conserved hypothetical proteins are designated by ORF length (in amino acid residues, where this is known). ORFs with significant homology to housekeeping genes are labeled with standard gene abbreviations. A truncated transposase coding sequence is indicated ( tnp ) by an open square. For ICEF-IA and ICEF-IIC, horizontal arrows indicate the relative positions and orientations of the primers used for PCR amplification of the corresponding sites in the chromosome of M. fermentans II-29/1 (which lacks ICEF units). The chromosomal region of strain II-29/1 that is equivalent to that occupied by ICEF-IIA in strain PG18 was analyzed by sequencing a cloned genomic fragment (see Materials and Methods). ICEF-IIB is inserted into one copy of ISMi 1 that is flanked by ISMf 1 and two hypothetical genes.
Figure Legend Snippet: Genomic locations of ICEF insertion sites in M. fermentans . The locations of ICEF insertion sites (solid arrows) in M. fermentans PG18 are shown, together with the equivalent region (for ICEF-IIA) of the strain II-29/1 chromosome. The locations and directions of ORFs (open arrows) and individual copies of multicopy ISs ISMi 1 and ISMf 1 (open rectangles) that flank each of the four ICEF units in M. fermentans PG18 are shown. ORFs for coding sequences that either lack homology to known proteins or encode homologs of conserved hypothetical proteins are designated by ORF length (in amino acid residues, where this is known). ORFs with significant homology to housekeeping genes are labeled with standard gene abbreviations. A truncated transposase coding sequence is indicated ( tnp ) by an open square. For ICEF-IA and ICEF-IIC, horizontal arrows indicate the relative positions and orientations of the primers used for PCR amplification of the corresponding sites in the chromosome of M. fermentans II-29/1 (which lacks ICEF units). The chromosomal region of strain II-29/1 that is equivalent to that occupied by ICEF-IIA in strain PG18 was analyzed by sequencing a cloned genomic fragment (see Materials and Methods). ICEF-IIB is inserted into one copy of ISMi 1 that is flanked by ISMf 1 and two hypothetical genes.

Techniques Used: Labeling, Sequencing, IA, Polymerase Chain Reaction, Amplification, Clone Assay

15) Product Images from "Transforming and Tumorigenic Activity of JAK2 by Fusion to BCR: Molecular Mechanisms of Action of a Novel BCR-JAK2 Tyrosine-Kinase"

Article Title: Transforming and Tumorigenic Activity of JAK2 by Fusion to BCR: Molecular Mechanisms of Action of a Novel BCR-JAK2 Tyrosine-Kinase

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032451

Cloning and identification of BCR-JAK2 fusion gene. ( a ) Nested RT-PCR analysis with standard primers for p190 BCR-ABL amplification (BCR-B and ABL3) on the ALL patient's samples at diagnosis (PB, peripheral blood; BM, bone marrow) showing an unexpected PCR product of 281 bp. PC: positive control (BCR-ABL + p190); NC, healthy negative control. ( b ) Verification of the BCR-JAK2 fusion gene by RT-PCR analysis using primers for BCR (BCR-B) and for the region flanking the stop codon of JAK2 (JAK2-Rv-T). NC, BCR-ABL + p210; MW, molecular weight marker. The 1,370-bp product contains part of BCR exon 1 fused to exon 19 of JAK2 tyrosine-kinase. ( c ) Full-length BCR-JAK2 cDNA amplification. RT-PCR with primers extending the start codon of BCR and the stop codon of JAK2 . ( d ) Schematic diagram of the putative BCR-JAK2 fusion protein (strips represent the fragment of the JAK2 domain JH2). Nucleotide (nt) and amino acid (aa) sequences below showed the BCR-JAK2 breakpoint region (red lettering represents the common BCR and JAK2 triplet). Numbers indicate the nucleotide breakpoint position: 1767 for the BCR gene (NM_004327) and 2929 for JAK2 (NM_004972). ( e ) Interphase nuclei of bone marrow cells at diagnosis after FISH analysis with a probe extending the 5′ region of BCR , showing three green signals indicating a translocation affecting one BCR allele. Schematic BCR gene structure and probe localization are represented below the nuclei. ( f ) FISH analysis with BAC probes flanking JAK2 . Co-localized green and red signals correspond to the normal JAK2 copy. Rearranged JAK2 allele is detected in the patient's nucleus as single green and red signals. ( g ) FISH analysis for BCR-JAK2 translocation. Two BCR (green) and two JAK2 (red) signals were observed in the control, whereas three BCR signals were detected in the patient, one of them fused with one red signal, revealing fusion between BCR and JAK2 . ( h ) BCR-JAK2 qPCR. Specific FRET-hybridization probes were used. Arbitrary values for BCR-JAK2 transcripts from diagnosis up to the present time are represented. Data were normalized with BCR . Molecular response was achieved before SCT. BM, bone marrow; PB, peripheral blood.
Figure Legend Snippet: Cloning and identification of BCR-JAK2 fusion gene. ( a ) Nested RT-PCR analysis with standard primers for p190 BCR-ABL amplification (BCR-B and ABL3) on the ALL patient's samples at diagnosis (PB, peripheral blood; BM, bone marrow) showing an unexpected PCR product of 281 bp. PC: positive control (BCR-ABL + p190); NC, healthy negative control. ( b ) Verification of the BCR-JAK2 fusion gene by RT-PCR analysis using primers for BCR (BCR-B) and for the region flanking the stop codon of JAK2 (JAK2-Rv-T). NC, BCR-ABL + p210; MW, molecular weight marker. The 1,370-bp product contains part of BCR exon 1 fused to exon 19 of JAK2 tyrosine-kinase. ( c ) Full-length BCR-JAK2 cDNA amplification. RT-PCR with primers extending the start codon of BCR and the stop codon of JAK2 . ( d ) Schematic diagram of the putative BCR-JAK2 fusion protein (strips represent the fragment of the JAK2 domain JH2). Nucleotide (nt) and amino acid (aa) sequences below showed the BCR-JAK2 breakpoint region (red lettering represents the common BCR and JAK2 triplet). Numbers indicate the nucleotide breakpoint position: 1767 for the BCR gene (NM_004327) and 2929 for JAK2 (NM_004972). ( e ) Interphase nuclei of bone marrow cells at diagnosis after FISH analysis with a probe extending the 5′ region of BCR , showing three green signals indicating a translocation affecting one BCR allele. Schematic BCR gene structure and probe localization are represented below the nuclei. ( f ) FISH analysis with BAC probes flanking JAK2 . Co-localized green and red signals correspond to the normal JAK2 copy. Rearranged JAK2 allele is detected in the patient's nucleus as single green and red signals. ( g ) FISH analysis for BCR-JAK2 translocation. Two BCR (green) and two JAK2 (red) signals were observed in the control, whereas three BCR signals were detected in the patient, one of them fused with one red signal, revealing fusion between BCR and JAK2 . ( h ) BCR-JAK2 qPCR. Specific FRET-hybridization probes were used. Arbitrary values for BCR-JAK2 transcripts from diagnosis up to the present time are represented. Data were normalized with BCR . Molecular response was achieved before SCT. BM, bone marrow; PB, peripheral blood.

Techniques Used: Clone Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Positive Control, Negative Control, Molecular Weight, Marker, Fluorescence In Situ Hybridization, Translocation Assay, BAC Assay, Real-time Polymerase Chain Reaction, Hybridization

Nude mice injected with Ba/F3-BCR-JAK2 cells develop tumors. Mice were subcutaneously injected with 10 7 Ba/F3-mock cells (left flank) and Ba/F3-BCR-JAK2 cells (right flank). ( a ) Left panel: white light photograph showing mice bearing a tumor only on the right side, where Ba/F3-BCR-JAK2 cells were injected (black arrows). Right panel: EGFP + tumor (white arrows) captured on a digital photostation showing the reflected image of the mice (right side). No EGFP expression was detected when Ba/F3-mock cells were injected (left side). ( b ) Flow cytometry analysis of EGFP-expressing tumor cells. ( c ) RT-PCR analysis to detect BCR-JAK2 expression using BCR-B and JAK2-3 primers. RNA from three different tumors was analyzed compared with RNA from Ba/F3-mock and Ba/F3-BCR-JAK2 cells used as negative and positive controls, respectively. pLZR-BCR-JAK2: plasmid bearing BCR-JAK2 was used as positive control. MW, marker.
Figure Legend Snippet: Nude mice injected with Ba/F3-BCR-JAK2 cells develop tumors. Mice were subcutaneously injected with 10 7 Ba/F3-mock cells (left flank) and Ba/F3-BCR-JAK2 cells (right flank). ( a ) Left panel: white light photograph showing mice bearing a tumor only on the right side, where Ba/F3-BCR-JAK2 cells were injected (black arrows). Right panel: EGFP + tumor (white arrows) captured on a digital photostation showing the reflected image of the mice (right side). No EGFP expression was detected when Ba/F3-mock cells were injected (left side). ( b ) Flow cytometry analysis of EGFP-expressing tumor cells. ( c ) RT-PCR analysis to detect BCR-JAK2 expression using BCR-B and JAK2-3 primers. RNA from three different tumors was analyzed compared with RNA from Ba/F3-mock and Ba/F3-BCR-JAK2 cells used as negative and positive controls, respectively. pLZR-BCR-JAK2: plasmid bearing BCR-JAK2 was used as positive control. MW, marker.

Techniques Used: Mouse Assay, Injection, Expressing, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Marker

16) Product Images from "Phase-defined complete sequencing of the HLA genes by next-generation sequencing"

Article Title: Phase-defined complete sequencing of the HLA genes by next-generation sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-355

Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.
Figure Legend Snippet: Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.

Techniques Used: Selection, Agarose Gel Electrophoresis, Sample Prep, Concentration Assay, Bridge PCR, Amplification

17) Product Images from "Tracing notochord-derived cells using a Noto-cre mouse: implications for intervertebral disc development"

Article Title: Tracing notochord-derived cells using a Noto-cre mouse: implications for intervertebral disc development

Journal: Disease Models & Mechanisms

doi: 10.1242/dmm.008128

Generation of the notochord-specific Cre mouse line. (A) Targeting strategy used to generate the Noto cre/+ line. An internal ribosome entry site-nuclear localization signal-Cre recombinase (IRES-NLS-CRE) cassette replaced exon 2 of the Noto locus. Positively targeted ES cell clones were confirmed by Southern blot using an external 5′ probe; the wild-type allele is 15 kb and the targeted allele is 10 kb. Representative positive ‘neo-in’ clones are shown. The positions of genotyping PCR primers for wild-type and ‘neo-out’ mice are also shown. (B) Targeting of the Noto locus does not affect its temporal regulation, as demonstrated by whole mount in situ hybridization at E11.5. Noto expression is detected in both Noto cre/+ and wild-type ( Noto +/+ ) littermate control embryos, localized to the posterior node in the tail region (insert and arrows). (C) Noto expression is downregulated after E12.5, confirmed by in situ hybridization at E15.5 showing no detectable Noto expression. (D) Heterozygous inactivation of Noto does not disrupt notochord formation or IVD development. IVD formation and tissue architecture was examined in Noto cre/+ mice and wild-type ( Noto +/+ ) littermate controls using Safranin-O/Fast Green staining on paraffin embedded sections at P21. Enlarged view of the NP and inner AF tissues are shown in the right-hand box. Scale bars: 500 μm for 100× images and 50 μm for 400× images.
Figure Legend Snippet: Generation of the notochord-specific Cre mouse line. (A) Targeting strategy used to generate the Noto cre/+ line. An internal ribosome entry site-nuclear localization signal-Cre recombinase (IRES-NLS-CRE) cassette replaced exon 2 of the Noto locus. Positively targeted ES cell clones were confirmed by Southern blot using an external 5′ probe; the wild-type allele is 15 kb and the targeted allele is 10 kb. Representative positive ‘neo-in’ clones are shown. The positions of genotyping PCR primers for wild-type and ‘neo-out’ mice are also shown. (B) Targeting of the Noto locus does not affect its temporal regulation, as demonstrated by whole mount in situ hybridization at E11.5. Noto expression is detected in both Noto cre/+ and wild-type ( Noto +/+ ) littermate control embryos, localized to the posterior node in the tail region (insert and arrows). (C) Noto expression is downregulated after E12.5, confirmed by in situ hybridization at E15.5 showing no detectable Noto expression. (D) Heterozygous inactivation of Noto does not disrupt notochord formation or IVD development. IVD formation and tissue architecture was examined in Noto cre/+ mice and wild-type ( Noto +/+ ) littermate controls using Safranin-O/Fast Green staining on paraffin embedded sections at P21. Enlarged view of the NP and inner AF tissues are shown in the right-hand box. Scale bars: 500 μm for 100× images and 50 μm for 400× images.

Techniques Used: Clone Assay, Southern Blot, Polymerase Chain Reaction, Mouse Assay, In Situ Hybridization, Expressing, Staining

18) Product Images from "SOD2 polymorphisms: unmasking the effect of polymorphism on splicing"

Article Title: SOD2 polymorphisms: unmasking the effect of polymorphism on splicing

Journal: BMC Medical Genetics

doi: 10.1186/1471-2350-8-7

Exon trapping assay of SOD2 intron 2 – exon 5 region. Ethidium bromide staining of the RT-PCR products from the exon trapping of the polymorphic genomic fragments (containing either T10 and T9 polymorphism). M is the marker lane with described sizes.
Figure Legend Snippet: Exon trapping assay of SOD2 intron 2 – exon 5 region. Ethidium bromide staining of the RT-PCR products from the exon trapping of the polymorphic genomic fragments (containing either T10 and T9 polymorphism). M is the marker lane with described sizes.

Techniques Used: Staining, Reverse Transcription Polymerase Chain Reaction, Marker

RT-PCR detection of SOD2 mRNAs. Primer sets were designed RT-PCR product spanning exon 2 through 5 (2–3–4–5), exon 3 through 5 (3–4–5), exon 4 to 5 (4–5) and exon 3 to 5 deleting 4 (3–5). Each set are tested in two T10/T10 homozygotes (first 2 lanes) and three T10/T9 heterozygotes (next 3 lanes) at three different annealing temperatures, 55°C, 58°C, and 60°C. Bottom panel shows the RT-PCR products with the primer sets that detect exon 4–5 only, 3–5 only and the combination of 4–5 plus 3–5.
Figure Legend Snippet: RT-PCR detection of SOD2 mRNAs. Primer sets were designed RT-PCR product spanning exon 2 through 5 (2–3–4–5), exon 3 through 5 (3–4–5), exon 4 to 5 (4–5) and exon 3 to 5 deleting 4 (3–5). Each set are tested in two T10/T10 homozygotes (first 2 lanes) and three T10/T9 heterozygotes (next 3 lanes) at three different annealing temperatures, 55°C, 58°C, and 60°C. Bottom panel shows the RT-PCR products with the primer sets that detect exon 4–5 only, 3–5 only and the combination of 4–5 plus 3–5.

Techniques Used: Reverse Transcription Polymerase Chain Reaction

19) Product Images from "Juxtaposition of heterochromatic and euchromatic regions by chromosomal translocation mediates a heterochromatic long-range position effect associated with a severe neurological phenotype"

Article Title: Juxtaposition of heterochromatic and euchromatic regions by chromosomal translocation mediates a heterochromatic long-range position effect associated with a severe neurological phenotype

Journal: Molecular Cytogenetics

doi: 10.1186/1755-8166-5-16

Real-time quantitative PCR showing the relative quantification (RQ) of ITFG1 cDNA amplification product obtained using ex-18 primers, which was found to be half that obtained using ex-3 primers . The expected ΔΔCt ratio is ≅1 when both alleles are expressed, and 0.5 when only one allele is expressed. The genes are indicated on the abscissa and the average of three recorded expression levels on the ordinate. The grey and white bars respectively represent the case and control.
Figure Legend Snippet: Real-time quantitative PCR showing the relative quantification (RQ) of ITFG1 cDNA amplification product obtained using ex-18 primers, which was found to be half that obtained using ex-3 primers . The expected ΔΔCt ratio is ≅1 when both alleles are expressed, and 0.5 when only one allele is expressed. The genes are indicated on the abscissa and the average of three recorded expression levels on the ordinate. The grey and white bars respectively represent the case and control.

Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Expressing

Related Articles

Sequencing:

Article Title: Phase-defined complete sequencing of the HLA genes by next-generation sequencing
Article Snippet: .. Most recently, 8-digit sequencing of HLA-genes is partially achieved using a combination of long-range PCR and Roche GS Junior sequencer and/or IonPGM sequencer [ ]. .. In their study, the closest HLA gene sequence from the IMGT/HLA database was selected as the reference sequence for alignment and phasing, and subsequently they could construct consensus sequence to call HLA alleles.

Polymerase Chain Reaction:

Article Title: Phase-defined complete sequencing of the HLA genes by next-generation sequencing
Article Snippet: .. Most recently, 8-digit sequencing of HLA-genes is partially achieved using a combination of long-range PCR and Roche GS Junior sequencer and/or IonPGM sequencer [ ]. .. In their study, the closest HLA gene sequence from the IMGT/HLA database was selected as the reference sequence for alignment and phasing, and subsequently they could construct consensus sequence to call HLA alleles.

Article Title: Phase-defined complete sequencing of the HLA genes by next-generation sequencing
Article Snippet: .. The six highly polymorphic HLA genes (HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1 ) were amplified by long-range PCR and the PCR amplicons covering full sequences of the genes were subjected to the MiSeq sequencer via the transposase-based library preparation. .. The derived paired-end reads (2 × 250 bp) from the MiSeq sequencer were analyzed by the one step alignments to the UCSC hg19 to obtain phase-defined complete sequences.

Article Title: Extensive Variation in Gene Copy Number at the Killer Immunoglobulin-Like Receptor Locus in Humans
Article Snippet: .. All KIR genes of 5 individuals were amplified by long-range PCR and sequenced by Next-generation sequencing (NGS) on a 454 FLX Roche Genome Sequencer to resolve these discrepancies ( ). .. In all cases, the methodological discrepancies appeared to be due to false-positive or false-negative PCR reactions in the PCR-SSP.

Article Title: High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic
Article Snippet: .. Confirmation and characterization of the rearrangements Positive results detected by MLPA of two independently drawn samples of genomic DNA were confirmed by long-range PCR (Expand Long Template PCR System, Roche Applied Science), conducted in accordance with the manufacturer's instructions. ..

Article Title: Mitochondrial DNA deletions and depletion within paraspinal muscles
Article Snippet: .. Fibres were lysed and long-range PCR was used to identify mtDNA deletions within the major arc of the mitochondrial genome using the Expand Long Template PCR System (Roche, Indianapolis, IN, USA), as previously described [ ]. .. DNA products were separated using a 0.7% agarose gel containing ethidium bromide and viewed under UV light.

Article Title: Phase-defined complete sequencing of the HLA genes by next-generation sequencing
Article Snippet: .. We applied long-range PCR to amplify six HLA genes (HLA-A, -C, -B, DRB1, -DQB1, and –DPB1 ) that are known to be highly polymorphic. .. PCR primers were designed to anneal where known polymorphic sites were not observed according to the dbSNP build 135 database, and to amplify regions spanning the promoter to 3′UTR of the HLA genes (Additional file : Table S1).

Article Title: Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression
Article Snippet: .. The entire fnr region was amplified by long-range PCR according to the manufacturer's instructions (Expand PCR Long Range; Boehringer-Roche) with 40 ng of genomic DNA, 3.75 U of Expand polymerase, 1× reaction buffer 2, 500 μM deoxynucleoside triphosphate mix, and 300 nM each primer in a 50-μl reaction) with forward primer b1331 and reverse primer b1345 at 94°C for 120 s and then for 10 cycles at 94°C for 10 s, 60°C for 30 s, and 68°C for 15 min, and 15 additional cycles with an increase of 20 s per cycle at 68°C (see Fig. ). ..

Article Title: A Gata4 nuclear GFP transcriptional reporter to study endoderm and cardiac development in the mouse
Article Snippet: .. Positive ES cells were identified by long-range PCR (Roche, Cat. 04829069001) with primer pair P1: 5′-TGGACGTGGACCACTGAGAGTAGG-3′ and P2: 5′-GCTTTAGTCACCGCCTTCTTGGAG-3′ and primer pair P3: 5′-CATGACATCGATTACAAGGATGACG-3′ and P4: 5′-CCTCAGTCTTCAACTCTCTGAACACC-3′. .. The PCR fragments were further verified by DNA sequencing.

Next-Generation Sequencing:

Article Title: Extensive Variation in Gene Copy Number at the Killer Immunoglobulin-Like Receptor Locus in Humans
Article Snippet: .. All KIR genes of 5 individuals were amplified by long-range PCR and sequenced by Next-generation sequencing (NGS) on a 454 FLX Roche Genome Sequencer to resolve these discrepancies ( ). .. In all cases, the methodological discrepancies appeared to be due to false-positive or false-negative PCR reactions in the PCR-SSP.

Amplification:

Article Title: Phase-defined complete sequencing of the HLA genes by next-generation sequencing
Article Snippet: .. The six highly polymorphic HLA genes (HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1 ) were amplified by long-range PCR and the PCR amplicons covering full sequences of the genes were subjected to the MiSeq sequencer via the transposase-based library preparation. .. The derived paired-end reads (2 × 250 bp) from the MiSeq sequencer were analyzed by the one step alignments to the UCSC hg19 to obtain phase-defined complete sequences.

Article Title: Extensive Variation in Gene Copy Number at the Killer Immunoglobulin-Like Receptor Locus in Humans
Article Snippet: .. All KIR genes of 5 individuals were amplified by long-range PCR and sequenced by Next-generation sequencing (NGS) on a 454 FLX Roche Genome Sequencer to resolve these discrepancies ( ). .. In all cases, the methodological discrepancies appeared to be due to false-positive or false-negative PCR reactions in the PCR-SSP.

Article Title: Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression
Article Snippet: .. The entire fnr region was amplified by long-range PCR according to the manufacturer's instructions (Expand PCR Long Range; Boehringer-Roche) with 40 ng of genomic DNA, 3.75 U of Expand polymerase, 1× reaction buffer 2, 500 μM deoxynucleoside triphosphate mix, and 300 nM each primer in a 50-μl reaction) with forward primer b1331 and reverse primer b1345 at 94°C for 120 s and then for 10 cycles at 94°C for 10 s, 60°C for 30 s, and 68°C for 15 min, and 15 additional cycles with an increase of 20 s per cycle at 68°C (see Fig. ). ..

Multiplex Ligation-dependent Probe Amplification:

Article Title: High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic
Article Snippet: .. Confirmation and characterization of the rearrangements Positive results detected by MLPA of two independently drawn samples of genomic DNA were confirmed by long-range PCR (Expand Long Template PCR System, Roche Applied Science), conducted in accordance with the manufacturer's instructions. ..

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    Roche long range pcr
    Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective <t>HLA</t> gene haplotype phasing. The size selection also determines an actual molar concentration for bridge <t>PCR</t> to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.
    Long Range Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 168 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche long range pcr analysis
    Relative copy number of LDL receptor gene exon 5 . Boxplots of relative copy number of LDL receptor gene exon 5 measured with Real-Time <t>PCR</t> Analysis and <t>MLPA</t> analysis showing median; box: 25 th -75 th percentile; bars: largest and smallest values within 1.5 box lengths; circles: outliers.
    Long Range Pcr Analysis, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 4 article reviews
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    Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.

    Journal: BMC Genomics

    Article Title: Phase-defined complete sequencing of the HLA genes by next-generation sequencing

    doi: 10.1186/1471-2164-14-355

    Figure Lengend Snippet: Size selection of the Nextera DNA libraries by agarose gel size selection. ( A ) Electropherogram of DNA library analyzed by 2100 Bioanalyzer. The library size of the Nextera DNA Sample Prep Kits was 150 bp to more than 10 kb (mean size: 902 bp). ( B ) Bioanalyzer electropherogram of a selected DNA library by cutting from the agarose gel. We selected large fragments with sizes ranging from 500 to 2,000 bp to remove short DNA fragments for effective HLA gene haplotype phasing. The size selection also determines an actual molar concentration for bridge PCR to generate clusters in flowcell, because DNA fragments with over 1.5 kb size are not efficiently amplified. The mean size of the selected fragments was 1,561 bp.

    Article Snippet: The six highly polymorphic HLA genes (HLA-A, -C, -B, -DRB1, -DQB1, and -DPB1 ) were amplified by long-range PCR and the PCR amplicons covering full sequences of the genes were subjected to the MiSeq sequencer via the transposase-based library preparation.

    Techniques: Selection, Agarose Gel Electrophoresis, Sample Prep, Concentration Assay, Bridge PCR, Amplification

    Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 1A/1B-2 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exons 5–14 by long-range PCR and sequencing of the breakpoints. (C) Confirmation of the deletion of exons 11–12 by long-range PCR and sequencing of the breakpoints. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Journal: BMC Medical Genetics

    Article Title: High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic

    doi: 10.1186/1471-2350-8-32

    Figure Lengend Snippet: Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 1A/1B-2 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exons 5–14 by long-range PCR and sequencing of the breakpoints. (C) Confirmation of the deletion of exons 11–12 by long-range PCR and sequencing of the breakpoints. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Article Snippet: Confirmation and characterization of the rearrangements Positive results detected by MLPA of two independently drawn samples of genomic DNA were confirmed by long-range PCR (Expand Long Template PCR System, Roche Applied Science), conducted in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

    Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 18–19 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exon 20 and sequencing of the breakpoints.Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Journal: BMC Medical Genetics

    Article Title: High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic

    doi: 10.1186/1471-2350-8-32

    Figure Lengend Snippet: Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 18–19 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exon 20 and sequencing of the breakpoints.Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Article Snippet: Confirmation and characterization of the rearrangements Positive results detected by MLPA of two independently drawn samples of genomic DNA were confirmed by long-range PCR (Expand Long Template PCR System, Roche Applied Science), conducted in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

    Confirmation and characterization of the rearrangements . Confirmation of the deletion of the exons 21–22 by long-range PCR and sequencing of the breakpoints. The deletion/insertion event was characterized as g.77128_80906del3779ins236. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Journal: BMC Medical Genetics

    Article Title: High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic

    doi: 10.1186/1471-2350-8-32

    Figure Lengend Snippet: Confirmation and characterization of the rearrangements . Confirmation of the deletion of the exons 21–22 by long-range PCR and sequencing of the breakpoints. The deletion/insertion event was characterized as g.77128_80906del3779ins236. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Article Snippet: Confirmation and characterization of the rearrangements Positive results detected by MLPA of two independently drawn samples of genomic DNA were confirmed by long-range PCR (Expand Long Template PCR System, Roche Applied Science), conducted in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

    LCT13 and TFPI-2as expression is linked. ( A ) Schematic diagram of the genomic region in Figure 1 A indicating regions (1–7) analysed by strand-specific RT–PCR (middle). Shown above and below the schematic are the ethidium bromide–stained gels used to visualize the strand-specific RT–PCR. Regions 2–7 are specifically expressed in cancer cell lines (H, HCC-1954 and M, MCF-7), but not normal breast (N), showing that cancer-specific antisense transcription is detectable up to 300 kb away from the TFPI-2 gene and up to the LINE-1 retrotransposon associated with LCT13. ( B ) siRNA knockdown of the LCT13 transcript. 2D densitometry of semiquantitative strand-specific RT–PCR analysis normalized to APRT control reveals an approximate 50% knockdown in LCT13 levels in cells transfected with a pool of three siRNA duplexes directed against LCT13 compared to those transfected with scrambled control siRNAs (left panel). This is paralleled by a 40–50% decrease in the TFPI-2as transcript (right panel).

    Journal: Nucleic Acids Research

    Article Title: Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer

    doi: 10.1093/nar/gkt438

    Figure Lengend Snippet: LCT13 and TFPI-2as expression is linked. ( A ) Schematic diagram of the genomic region in Figure 1 A indicating regions (1–7) analysed by strand-specific RT–PCR (middle). Shown above and below the schematic are the ethidium bromide–stained gels used to visualize the strand-specific RT–PCR. Regions 2–7 are specifically expressed in cancer cell lines (H, HCC-1954 and M, MCF-7), but not normal breast (N), showing that cancer-specific antisense transcription is detectable up to 300 kb away from the TFPI-2 gene and up to the LINE-1 retrotransposon associated with LCT13. ( B ) siRNA knockdown of the LCT13 transcript. 2D densitometry of semiquantitative strand-specific RT–PCR analysis normalized to APRT control reveals an approximate 50% knockdown in LCT13 levels in cells transfected with a pool of three siRNA duplexes directed against LCT13 compared to those transfected with scrambled control siRNAs (left panel). This is paralleled by a 40–50% decrease in the TFPI-2as transcript (right panel).

    Article Snippet: Generation of constructs and ES cell clones For pTFPI-2as and pTFPI-2pa constructs, a 4.93-kb human genomic DNA fragment including the full-length TFPI-2 gene obtained by long-range PCR (Expand Long PCR kit, Roche) on human genomic DNA with primers HC63f and HC63g and was cloned into the BamHI and KpnI sites of pcDNA3 (Invitrogen) and pcDNA3p(A)for, respectively. pcDNA3p(A)for was derived from pcDNA3 by cloning a 262 bp BGHp(A) fragment, obtained by PCR on pcDNA3 with primers Hind-p(A)-for and Hind-p(A)-rev, into the HindIII site of pcDNA3.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Transfection

    A human TFPI-2 transgene is sensitive to antisense RNA repression in mouse ES cells. (A) Schematic diagram of constructs introduced into mouse ES cells: pTFPI-2as is designed to transcribe antisense to TFPI-2 from a CMV promoter, while pTFPI-2pa has a poly-A signal insertion downstream of the CMV promoter to block antisense transcription. Arrows indicate direction of transcription. Regions analysed by ChIP are annotated as ‘prom’ and ‘ex-in2’. ( B ) Strand-specific RT–PCR analysis of TFPI-2 antisenese (TFPI-2as) expression in transgenic mouse ES cell lines demonstrates increased levels in pTFPI-2as lines (L2 and L12) relative to pTFPI-2pa cells (L7 and L9), mouse Aprt acts as a positive control for RNA quality and quantity. This correlates with a reduction in TFPI-2 expression as shown by real-time PCR normalized to mouse Gapdh . ( C ) ChIP analysis followed by real-time PCR. Left panel: Antibodies to H3K9me3 reveal localized enrichment of H3K9me3 in the promoter region in the antisense expressing cell line, pTFPI-2as (L2), compared to cells transfected with pTFPI-2pa (L9), which express low levels of TFPI-2as. Right panel: Antibodies to H4K20me3 also show enrichment at the TFPI-2 promoter in pTFPI-2as compared to pTFPI-2pa.

    Journal: Nucleic Acids Research

    Article Title: Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer

    doi: 10.1093/nar/gkt438

    Figure Lengend Snippet: A human TFPI-2 transgene is sensitive to antisense RNA repression in mouse ES cells. (A) Schematic diagram of constructs introduced into mouse ES cells: pTFPI-2as is designed to transcribe antisense to TFPI-2 from a CMV promoter, while pTFPI-2pa has a poly-A signal insertion downstream of the CMV promoter to block antisense transcription. Arrows indicate direction of transcription. Regions analysed by ChIP are annotated as ‘prom’ and ‘ex-in2’. ( B ) Strand-specific RT–PCR analysis of TFPI-2 antisenese (TFPI-2as) expression in transgenic mouse ES cell lines demonstrates increased levels in pTFPI-2as lines (L2 and L12) relative to pTFPI-2pa cells (L7 and L9), mouse Aprt acts as a positive control for RNA quality and quantity. This correlates with a reduction in TFPI-2 expression as shown by real-time PCR normalized to mouse Gapdh . ( C ) ChIP analysis followed by real-time PCR. Left panel: Antibodies to H3K9me3 reveal localized enrichment of H3K9me3 in the promoter region in the antisense expressing cell line, pTFPI-2as (L2), compared to cells transfected with pTFPI-2pa (L9), which express low levels of TFPI-2as. Right panel: Antibodies to H4K20me3 also show enrichment at the TFPI-2 promoter in pTFPI-2as compared to pTFPI-2pa.

    Article Snippet: Generation of constructs and ES cell clones For pTFPI-2as and pTFPI-2pa constructs, a 4.93-kb human genomic DNA fragment including the full-length TFPI-2 gene obtained by long-range PCR (Expand Long PCR kit, Roche) on human genomic DNA with primers HC63f and HC63g and was cloned into the BamHI and KpnI sites of pcDNA3 (Invitrogen) and pcDNA3p(A)for, respectively. pcDNA3p(A)for was derived from pcDNA3 by cloning a 262 bp BGHp(A) fragment, obtained by PCR on pcDNA3 with primers Hind-p(A)-for and Hind-p(A)-rev, into the HindIII site of pcDNA3.

    Techniques: Construct, Blocking Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Positive Control, Real-time Polymerase Chain Reaction, Transfection

    Correlated expression of LCT13 and TFPI-2as transcripts in breast cancer cells. ( A ) Schematic diagram of a 300-kb region of chromosome 7q21.3 including LCT13 and the TFPI-2 gene. Scale is kilobase and indicates the position from the centromere with the value of 0 arbitrarily assigned to the TSS of CALCR . Genes (5′ segment of CALCR , TFPI-2 and GNGT1 ) are indicated as gray arrows. Two LINE-1 elements are present in the region (L1PA2 and L1PA6). Transcriptional orientations are indicated by arrows. LCT13 is a previously identified transcript shown to initiate at an L1ASP by 5′ RACE ( 22 ). TFPI-2as is the fragment analysed by strand-specific RT–PCR to test for the presence of TFPI-2 antisense RNAs. Displayed are the three spliced ESTs isolated from kidney (BG432114) and liver (DW466562 and DW435092) libraries that initiate at the LINE1 antisense promoter like LCT13 and extend past the TFPI-2 gene with a putative alternative transcript GNGT1-005 also annotated. ( B ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in normal breast (N) and in breast cancer cell lines (H, HCC-1954; M, MCF7) analysed by strand specific and real-time RT–PCR, respectively. TFPI-2 expression is reduced in both breast cancer cell lines compared to normal controls (n = 3). TFPI-2 expression levels were normalized to HPRT . ( C ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in a panel of five matched normal and tumour breast tissue analysed as described in B.

    Journal: Nucleic Acids Research

    Article Title: Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer

    doi: 10.1093/nar/gkt438

    Figure Lengend Snippet: Correlated expression of LCT13 and TFPI-2as transcripts in breast cancer cells. ( A ) Schematic diagram of a 300-kb region of chromosome 7q21.3 including LCT13 and the TFPI-2 gene. Scale is kilobase and indicates the position from the centromere with the value of 0 arbitrarily assigned to the TSS of CALCR . Genes (5′ segment of CALCR , TFPI-2 and GNGT1 ) are indicated as gray arrows. Two LINE-1 elements are present in the region (L1PA2 and L1PA6). Transcriptional orientations are indicated by arrows. LCT13 is a previously identified transcript shown to initiate at an L1ASP by 5′ RACE ( 22 ). TFPI-2as is the fragment analysed by strand-specific RT–PCR to test for the presence of TFPI-2 antisense RNAs. Displayed are the three spliced ESTs isolated from kidney (BG432114) and liver (DW466562 and DW435092) libraries that initiate at the LINE1 antisense promoter like LCT13 and extend past the TFPI-2 gene with a putative alternative transcript GNGT1-005 also annotated. ( B ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in normal breast (N) and in breast cancer cell lines (H, HCC-1954; M, MCF7) analysed by strand specific and real-time RT–PCR, respectively. TFPI-2 expression is reduced in both breast cancer cell lines compared to normal controls (n = 3). TFPI-2 expression levels were normalized to HPRT . ( C ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in a panel of five matched normal and tumour breast tissue analysed as described in B.

    Article Snippet: Generation of constructs and ES cell clones For pTFPI-2as and pTFPI-2pa constructs, a 4.93-kb human genomic DNA fragment including the full-length TFPI-2 gene obtained by long-range PCR (Expand Long PCR kit, Roche) on human genomic DNA with primers HC63f and HC63g and was cloned into the BamHI and KpnI sites of pcDNA3 (Invitrogen) and pcDNA3p(A)for, respectively. pcDNA3p(A)for was derived from pcDNA3 by cloning a 262 bp BGHp(A) fragment, obtained by PCR on pcDNA3 with primers Hind-p(A)-for and Hind-p(A)-rev, into the HindIII site of pcDNA3.

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

    Relative copy number of LDL receptor gene exon 5 . Boxplots of relative copy number of LDL receptor gene exon 5 measured with Real-Time PCR Analysis and MLPA analysis showing median; box: 25 th -75 th percentile; bars: largest and smallest values within 1.5 box lengths; circles: outliers.

    Journal: BMC Medical Genetics

    Article Title: Detection of large deletions in the LDL receptor gene with quantitative PCR methods

    doi: 10.1186/1471-2350-6-15

    Figure Lengend Snippet: Relative copy number of LDL receptor gene exon 5 . Boxplots of relative copy number of LDL receptor gene exon 5 measured with Real-Time PCR Analysis and MLPA analysis showing median; box: 25 th -75 th percentile; bars: largest and smallest values within 1.5 box lengths; circles: outliers.

    Article Snippet: Long-range PCR analysis When the results of MLPA analysis suggested that a major structural rearrangement was present in the LDL receptor gene, the results were confirmed with long-range PCR analysis [ ] using the Expand 20 kbPLUS PCR System (Roche).

    Techniques: Real-time Polymerase Chain Reaction, Multiplex Ligation-dependent Probe Amplification