Structured Review

MACHEREY NAGEL pcr clean up columns
Characterization of a stable <t>eys</t> rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of <t>RT-PCR</t> analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.
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Images

1) Product Images from "Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish"

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200789

Characterization of a stable eys rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.
Figure Legend Snippet: Characterization of a stable eys rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.

Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction

2) Product Images from "Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells"

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells

Journal: Cell metabolism

doi: 10.1016/j.cmet.2018.10.014

mtDNA Is Replenished and Respiration Recovers Early in Tumor Formation by 4T1ρ° Cells (A) BALB/c mice (n = 6) were grafted subcutaneously (s.c.) with 4T1 or 4T1 ρ° cells at 10 6 per animal, and tumor volume was assessed by ultrasound imaging (USI) (n = 6). (B and C) Time schedule of retrieval of pre-tumor plaques and tumors from BALB/c mice is shown in (B), as indicated in (C) for D5 tissue. (D) Individual lines retrieved from mice according to the schedule in (B) were assessed for uncoupled (ETC), routine, and leak respiration using the Oxygraph (n = 3). (E) Cell lines were evaluated for the presence of mtDNA using probes against a polymorphism in either 4T1 or host 16S rRNA by sc/qPCR; 92 cells per line were assessed. (F) Distribution of mtDNA polymorphism in 16S rRNA of individual cells of lines plotted using data presented in (E). (G) mTRIP assay in the presence of proteinase K was used to detect the mtDNA initiation of replication marker (mREP) and global mitochondrial transcripts (mTRANS) unmasked from proteins, in single cells (n = 3; 100 cells were assessed per condition). (H) Cell lines were assessed for binding of TFAM and POLG1 to the D LOOP region of mtDNA using a mitoChIP assay (n = 3). (I) Individual lines were probed for the level of mtDNA-processing proteins using WB. (J–L) qRT-PCR was used to assess the level of transcripts of selected subunits of mitochondrially encoded transcripts of RCs (n = 3) (J). Selected subunits of mitochondrial RCs were evaluated using WB in individual lines (K), which were then assessed by NBGE for the assembly of RCs and SCs using antibodies to relevant subunits, as shown (L). Data in (A), (D), (G), (H), and (J) are mean values ± SD. Representative images of three biological replicates are shown for (I), (K), and (L).
Figure Legend Snippet: mtDNA Is Replenished and Respiration Recovers Early in Tumor Formation by 4T1ρ° Cells (A) BALB/c mice (n = 6) were grafted subcutaneously (s.c.) with 4T1 or 4T1 ρ° cells at 10 6 per animal, and tumor volume was assessed by ultrasound imaging (USI) (n = 6). (B and C) Time schedule of retrieval of pre-tumor plaques and tumors from BALB/c mice is shown in (B), as indicated in (C) for D5 tissue. (D) Individual lines retrieved from mice according to the schedule in (B) were assessed for uncoupled (ETC), routine, and leak respiration using the Oxygraph (n = 3). (E) Cell lines were evaluated for the presence of mtDNA using probes against a polymorphism in either 4T1 or host 16S rRNA by sc/qPCR; 92 cells per line were assessed. (F) Distribution of mtDNA polymorphism in 16S rRNA of individual cells of lines plotted using data presented in (E). (G) mTRIP assay in the presence of proteinase K was used to detect the mtDNA initiation of replication marker (mREP) and global mitochondrial transcripts (mTRANS) unmasked from proteins, in single cells (n = 3; 100 cells were assessed per condition). (H) Cell lines were assessed for binding of TFAM and POLG1 to the D LOOP region of mtDNA using a mitoChIP assay (n = 3). (I) Individual lines were probed for the level of mtDNA-processing proteins using WB. (J–L) qRT-PCR was used to assess the level of transcripts of selected subunits of mitochondrially encoded transcripts of RCs (n = 3) (J). Selected subunits of mitochondrial RCs were evaluated using WB in individual lines (K), which were then assessed by NBGE for the assembly of RCs and SCs using antibodies to relevant subunits, as shown (L). Data in (A), (D), (G), (H), and (J) are mean values ± SD. Representative images of three biological replicates are shown for (I), (K), and (L).

Techniques Used: Mouse Assay, Imaging, Real-time Polymerase Chain Reaction, Marker, Binding Assay, Western Blot, Quantitative RT-PCR

Respiration Recovery Is Associated with Reactivation of DHODH (A) Cell lines as shown were evaluated for growth in the presence of uridine and pyruvate, or with pyruvate or uridine removed. (B) Cell lines were assessed for DHODH expression by qRT-PCR and WB. (C–E) DHODH-dependent respiration was assessed in parental 4T1 cells in the absence and presence of 30 μM leflunomide (Lef) (C). Individual lines were assessed for DHODH-dependent respiration (D) and for the orotate-to-DHO ratio (E). (F) Parental, D0, D5, D10, D15, D20, D25, and D60 cells (10 6 ) were grafted s.c. into BALB/c mice, and tumor growth was evaluated by USI. (G) Tumors grown from parental or 4T1 ρ° cells were excised and processed in a dedicated tissue shredder, and the homogenates were assessed for CI-, CII-, and DHODH-dependent respiration using an Oxygraph. (H) BALB/c mice were grafted s.c. with 10 6 D0 cells. On the days indicated, animals were sacrificed and tissue from the grafted region excised, sectioned, and assessed by immunohistochemistry for proliferation using anti-Ki67 IgG followed by confocal microscopy. The numbers indicate percentage of Ki67-positive cells. Data in (A) are mean values derived from two biological replicates with differences less than 10%; data in (B) are mean values ±SD (n ≥ 3); data in (C–G) are mean values ± SEM (n = 3); images in (B) and (H) are representative of three independent experiments. The symbol “*” indicates statistically significant differences from parental cells, and the symbol “#” indicates statistically significant difference from D0 cells, with p
Figure Legend Snippet: Respiration Recovery Is Associated with Reactivation of DHODH (A) Cell lines as shown were evaluated for growth in the presence of uridine and pyruvate, or with pyruvate or uridine removed. (B) Cell lines were assessed for DHODH expression by qRT-PCR and WB. (C–E) DHODH-dependent respiration was assessed in parental 4T1 cells in the absence and presence of 30 μM leflunomide (Lef) (C). Individual lines were assessed for DHODH-dependent respiration (D) and for the orotate-to-DHO ratio (E). (F) Parental, D0, D5, D10, D15, D20, D25, and D60 cells (10 6 ) were grafted s.c. into BALB/c mice, and tumor growth was evaluated by USI. (G) Tumors grown from parental or 4T1 ρ° cells were excised and processed in a dedicated tissue shredder, and the homogenates were assessed for CI-, CII-, and DHODH-dependent respiration using an Oxygraph. (H) BALB/c mice were grafted s.c. with 10 6 D0 cells. On the days indicated, animals were sacrificed and tissue from the grafted region excised, sectioned, and assessed by immunohistochemistry for proliferation using anti-Ki67 IgG followed by confocal microscopy. The numbers indicate percentage of Ki67-positive cells. Data in (A) are mean values derived from two biological replicates with differences less than 10%; data in (B) are mean values ±SD (n ≥ 3); data in (C–G) are mean values ± SEM (n = 3); images in (B) and (H) are representative of three independent experiments. The symbol “*” indicates statistically significant differences from parental cells, and the symbol “#” indicates statistically significant difference from D0 cells, with p

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Mouse Assay, Immunohistochemistry, Confocal Microscopy, Derivative Assay

3) Product Images from "NG2 expression in NG2 glia is regulated by binding of SoxE and bHLH transcription factors to a Cspg4 intronic enhancer"

Article Title: NG2 expression in NG2 glia is regulated by binding of SoxE and bHLH transcription factors to a Cspg4 intronic enhancer

Journal: Glia

doi: 10.1002/glia.23521

ChIP to show binding of Olig2 and Sox10 and active histone marks at int1–3b. (A) Scheme of the Cspg4 gene showing the location of int1–3b and intron2 primers at the top. Arrows indicate primers used for ChIP-PCR. The image of a gel below shows PCR detection of int1–3b or intron 2 regions on immunoprecipitates formed from Oli-neu cell nuclear extracts using control rabbit IgG, rabbit anti-histone H3 antibody (positive control), rabbit anti-Olig2 antibody, and rabbit anti-Sox10 antibody. The expected size of int1–3b PCR product is 126 bp and is indicated by an arrowhead on the left margin. The expected size of intron 2 PCR product is 136 bp and is indicated by an arrowhead on the right margin. The positions of 200 bp and 100 bp on the DNA ladder are indicated by arrows. The faster migrating band across all lanes at the bottom indicated by an asterisk are likely to be primer dimers. (B) Genome browser tracks at the int1–3b and int2 loci showing ChIP-seq tracks from epiblast stem cell-derived OPCs. H3K4me1 and H3K27ac enhancer-associated histone modifications are enriched at int1–3b, but not at int2.
Figure Legend Snippet: ChIP to show binding of Olig2 and Sox10 and active histone marks at int1–3b. (A) Scheme of the Cspg4 gene showing the location of int1–3b and intron2 primers at the top. Arrows indicate primers used for ChIP-PCR. The image of a gel below shows PCR detection of int1–3b or intron 2 regions on immunoprecipitates formed from Oli-neu cell nuclear extracts using control rabbit IgG, rabbit anti-histone H3 antibody (positive control), rabbit anti-Olig2 antibody, and rabbit anti-Sox10 antibody. The expected size of int1–3b PCR product is 126 bp and is indicated by an arrowhead on the left margin. The expected size of intron 2 PCR product is 136 bp and is indicated by an arrowhead on the right margin. The positions of 200 bp and 100 bp on the DNA ladder are indicated by arrows. The faster migrating band across all lanes at the bottom indicated by an asterisk are likely to be primer dimers. (B) Genome browser tracks at the int1–3b and int2 loci showing ChIP-seq tracks from epiblast stem cell-derived OPCs. H3K4me1 and H3K27ac enhancer-associated histone modifications are enriched at int1–3b, but not at int2.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Positive Control, Derivative Assay

4) Product Images from "NG2 expression in NG2 glia is regulated by binding of SoxE and bHLH transcription factors to a Cspg4 intronic enhancer"

Article Title: NG2 expression in NG2 glia is regulated by binding of SoxE and bHLH transcription factors to a Cspg4 intronic enhancer

Journal: Glia

doi: 10.1002/glia.23521

ChIP to show binding of Olig2 and Sox10 and active histone marks at int1–3b. (A) Scheme of the Cspg4 gene showing the location of int1–3b and intron2 primers at the top. Arrows indicate primers used for ChIP-PCR. The image of a gel below shows PCR detection of int1–3b or intron 2 regions on immunoprecipitates formed from Oli-neu cell nuclear extracts using control rabbit IgG, rabbit anti-histone H3 antibody (positive control), rabbit anti-Olig2 antibody, and rabbit anti-Sox10 antibody. The expected size of int1–3b PCR product is 126 bp and is indicated by an arrowhead on the left margin. The expected size of intron 2 PCR product is 136 bp and is indicated by an arrowhead on the right margin. The positions of 200 bp and 100 bp on the DNA ladder are indicated by arrows. The faster migrating band across all lanes at the bottom indicated by an asterisk are likely to be primer dimers. (B) Genome browser tracks at the int1–3b and int2 loci showing ChIP-seq tracks from epiblast stem cell-derived OPCs. H3K4me1 and H3K27ac enhancer-associated histone modifications are enriched at int1–3b, but not at int2.
Figure Legend Snippet: ChIP to show binding of Olig2 and Sox10 and active histone marks at int1–3b. (A) Scheme of the Cspg4 gene showing the location of int1–3b and intron2 primers at the top. Arrows indicate primers used for ChIP-PCR. The image of a gel below shows PCR detection of int1–3b or intron 2 regions on immunoprecipitates formed from Oli-neu cell nuclear extracts using control rabbit IgG, rabbit anti-histone H3 antibody (positive control), rabbit anti-Olig2 antibody, and rabbit anti-Sox10 antibody. The expected size of int1–3b PCR product is 126 bp and is indicated by an arrowhead on the left margin. The expected size of intron 2 PCR product is 136 bp and is indicated by an arrowhead on the right margin. The positions of 200 bp and 100 bp on the DNA ladder are indicated by arrows. The faster migrating band across all lanes at the bottom indicated by an asterisk are likely to be primer dimers. (B) Genome browser tracks at the int1–3b and int2 loci showing ChIP-seq tracks from epiblast stem cell-derived OPCs. H3K4me1 and H3K27ac enhancer-associated histone modifications are enriched at int1–3b, but not at int2.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Positive Control, Derivative Assay

5) Product Images from "Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish"

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish

Journal: PLoS ONE

doi: 10.1371/journal.pone.0200789

Characterization of a stable eys rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.
Figure Legend Snippet: Characterization of a stable eys rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.

Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction

6) Product Images from "Introns Regulate Gene Expression in Cryptococcus neoformans in a Pab2p Dependent Pathway"

Article Title: Introns Regulate Gene Expression in Cryptococcus neoformans in a Pab2p Dependent Pathway

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003686

Influence of introns on gene expression. A . Schematic representation of the different cDNA molecules sequenced illustrating intron retention in CAS3 mRNA. CAS3 specific cDNA molecules were RT-PCR-amplified from purified mRNA, separated in three pools according to their sizes (Small, Medium and Large) and cloned. Fifteen clones were sequenced per pool and the presence of introns was analyzed. The numbers of clones obtained for each isoform in each pool are indicated. Introns and exons are not at real scale. B . cas3Δi is a non-functional allele. When the cas3Δ allele was replaced by the intronless allele, there were very little CAS3 mRNAs and no complementation of the capsule phenotype. (1) Antibody reactivity of the GXM-specific Mab CRND-8 with the mutant strains. 10 4 cells were spotted on a nitrocellulose membrane and probed with CRND-8. (2) Northern blot experiment results showing very low mRNA level of the cas3Δi allele. C . Intron-dependent regulation of mRNA accumulation is gene specific. Two independently obtained Δi strains were tested here giving identical results (data not shown). As a control, we checked that replacing the cas3Δ allele by the wild type gene ( CAS3 ) using the same procedure restored the antibody reactivity and the level of mRNA accumulation.
Figure Legend Snippet: Influence of introns on gene expression. A . Schematic representation of the different cDNA molecules sequenced illustrating intron retention in CAS3 mRNA. CAS3 specific cDNA molecules were RT-PCR-amplified from purified mRNA, separated in three pools according to their sizes (Small, Medium and Large) and cloned. Fifteen clones were sequenced per pool and the presence of introns was analyzed. The numbers of clones obtained for each isoform in each pool are indicated. Introns and exons are not at real scale. B . cas3Δi is a non-functional allele. When the cas3Δ allele was replaced by the intronless allele, there were very little CAS3 mRNAs and no complementation of the capsule phenotype. (1) Antibody reactivity of the GXM-specific Mab CRND-8 with the mutant strains. 10 4 cells were spotted on a nitrocellulose membrane and probed with CRND-8. (2) Northern blot experiment results showing very low mRNA level of the cas3Δi allele. C . Intron-dependent regulation of mRNA accumulation is gene specific. Two independently obtained Δi strains were tested here giving identical results (data not shown). As a control, we checked that replacing the cas3Δ allele by the wild type gene ( CAS3 ) using the same procedure restored the antibody reactivity and the level of mRNA accumulation.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Purification, Clone Assay, Functional Assay, Mutagenesis, Northern Blot

Xrn2p degrades cas3Δi mRNA in a Pab2p-dependent fashion. A . Simple depletion of XRN2 does not lead to cas3Δi mRNA stabilisation. Cells from different mutant strains grown overnight in galactose were washed and transferred to glucose for 10 h. RNA was extracted and 5 µg were loaded on a denaturing electrophoresis agarose gel, transferred on a nylon membrane and probed with CAS3 and XRN2 specific probes. B . Depletion of XRN2 leads to increased expression of RRP44 and vice versa . Cells from different mutant strains grown overnight in galactose were washed and transferred to glucose for 10 h. RNA was extracted and 5 µg were loaded on a denaturing electrophoresis agarose gel, transferred on a nylon membrane and probed with RRP44 and XRN2 specific probes. C. and D . The degradation of cas3Δi mRNA by Rrp44p is largely independent of Pab2p whereas that by Xrn2p rather depends on Pab2p. RNA was extracted from cells growing in YPG (5·10 7 cells/mL). 5 µg were loaded on a denaturing electrophoresis agarose gel, transferred on a nylon membrane and probed with CAS3 , RRP44 and XRN2 specific probes C. RNA was treated with Dnase I and 1 µg were used to synthesise cDNA. Each quantitative PCR run was assayed in triplicate. For qPCR analyses primers were chosen that amplify 131 bp of the 5′ part of the cas3Δi transcript ensuring the capture of all isoforms of cas3Δi mRNA stabilised upon PAB2 deletion. The reported values are the means ± SD of three independent experiments.
Figure Legend Snippet: Xrn2p degrades cas3Δi mRNA in a Pab2p-dependent fashion. A . Simple depletion of XRN2 does not lead to cas3Δi mRNA stabilisation. Cells from different mutant strains grown overnight in galactose were washed and transferred to glucose for 10 h. RNA was extracted and 5 µg were loaded on a denaturing electrophoresis agarose gel, transferred on a nylon membrane and probed with CAS3 and XRN2 specific probes. B . Depletion of XRN2 leads to increased expression of RRP44 and vice versa . Cells from different mutant strains grown overnight in galactose were washed and transferred to glucose for 10 h. RNA was extracted and 5 µg were loaded on a denaturing electrophoresis agarose gel, transferred on a nylon membrane and probed with RRP44 and XRN2 specific probes. C. and D . The degradation of cas3Δi mRNA by Rrp44p is largely independent of Pab2p whereas that by Xrn2p rather depends on Pab2p. RNA was extracted from cells growing in YPG (5·10 7 cells/mL). 5 µg were loaded on a denaturing electrophoresis agarose gel, transferred on a nylon membrane and probed with CAS3 , RRP44 and XRN2 specific probes C. RNA was treated with Dnase I and 1 µg were used to synthesise cDNA. Each quantitative PCR run was assayed in triplicate. For qPCR analyses primers were chosen that amplify 131 bp of the 5′ part of the cas3Δi transcript ensuring the capture of all isoforms of cas3Δi mRNA stabilised upon PAB2 deletion. The reported values are the means ± SD of three independent experiments.

Techniques Used: Mutagenesis, Electrophoresis, Agarose Gel Electrophoresis, Expressing, Real-time Polymerase Chain Reaction

Related Articles

Countercurrent Chromatography:

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells.
Article Snippet: .. After extensive washing, chromatin was eluted and treated with RNAseA and proteinase K to reverse the crosslinking, followed by purification using PCR clean-up columns (MachereyNagel). qRT-PCRwas then run using the Eva Green system (Solis Biodyne) at 95 C for 12min, 40 cycles of 95 C for 15 s, 60 C for 20 s, 72 C for 35 s, followed by melt curve analysis using primers for mouse mtDNA encompassing D-loop; the binding site of POLG: mPOLG ChIP F, TGA TCA ATT CTA GTA GTT CCC AAA A; mPOLG ChIP R, ACC TCT AAT TAA TTA TAA GGC CAGG. ..

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells
Article Snippet: .. After extensive washing, chromatin was eluted and treated with RNAseA and proteinase K to reverse the crosslinking, followed by purification using PCR clean-up columns (MachereyNagel). qRT-PCR was then run using the Eva Green system (Solis Biodyne) at 95°C for 12 min, 40 cycles of 95°C for 15 s, 60°C for 20 s, 72°C for 35 s, followed by melt curve analysis using primers for mouse mtDNA encompassing D-loop; the binding site of POLG: mPOLG ChIP F, TGA TCA ATT CTA GTA GTT CCC AAA A; mPOLG ChIP R, ACC TCT AAT TAA TTA TAA GGC CAGG. ..

Clone Assay:

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

Amplification:

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

Agarose Gel Electrophoresis:

Article Title: In cell mutational interference mapping experiment (in cell MIME) identifies the 5′ polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging
Article Snippet: .. Samples were then run on a 1% agarose gel and the range corresponding to 200–600 bp range was isolated, and purified using Nucleospin Gel and PCR Clean-up columns. .. DNA libraries were quantified by qPCR using Illumina PE PCR primer 1.0 and one of the Illumina Index primers (for multiplexing) with Brilliant II SYBR master mix.

Isolation:

Article Title: In cell mutational interference mapping experiment (in cell MIME) identifies the 5′ polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging
Article Snippet: .. Samples were then run on a 1% agarose gel and the range corresponding to 200–600 bp range was isolated, and purified using Nucleospin Gel and PCR Clean-up columns. .. DNA libraries were quantified by qPCR using Illumina PE PCR primer 1.0 and one of the Illumina Index primers (for multiplexing) with Brilliant II SYBR master mix.

TA Cloning:

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

Quantitative RT-PCR:

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells
Article Snippet: .. After extensive washing, chromatin was eluted and treated with RNAseA and proteinase K to reverse the crosslinking, followed by purification using PCR clean-up columns (MachereyNagel). qRT-PCR was then run using the Eva Green system (Solis Biodyne) at 95°C for 12 min, 40 cycles of 95°C for 15 s, 60°C for 20 s, 72°C for 35 s, followed by melt curve analysis using primers for mouse mtDNA encompassing D-loop; the binding site of POLG: mPOLG ChIP F, TGA TCA ATT CTA GTA GTT CCC AAA A; mPOLG ChIP R, ACC TCT AAT TAA TTA TAA GGC CAGG. ..

Purification:

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells.
Article Snippet: .. After extensive washing, chromatin was eluted and treated with RNAseA and proteinase K to reverse the crosslinking, followed by purification using PCR clean-up columns (MachereyNagel). qRT-PCRwas then run using the Eva Green system (Solis Biodyne) at 95 C for 12min, 40 cycles of 95 C for 15 s, 60 C for 20 s, 72 C for 35 s, followed by melt curve analysis using primers for mouse mtDNA encompassing D-loop; the binding site of POLG: mPOLG ChIP F, TGA TCA ATT CTA GTA GTT CCC AAA A; mPOLG ChIP R, ACC TCT AAT TAA TTA TAA GGC CAGG. ..

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

Article Title: NG2 expression in NG2 glia is regulated by binding of SoxE and bHLH transcription factors to a Cspg4 intronic enhancer
Article Snippet: .. Cross-links were reversed by incubating the eluted material in proteinase K at 65o C for 2 hours, and DNA was purified using NucleoSpin Gel and PCR Clean-up columns (Macherey-Nagel, USA). .. The purified DNA was subjected to PCR using int1–3b and intron 2 primers from the mouse Cspg4 gene ( ) and the products were separated on 1.8% agarose gels.

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells
Article Snippet: .. After extensive washing, chromatin was eluted and treated with RNAseA and proteinase K to reverse the crosslinking, followed by purification using PCR clean-up columns (MachereyNagel). qRT-PCR was then run using the Eva Green system (Solis Biodyne) at 95°C for 12 min, 40 cycles of 95°C for 15 s, 60°C for 20 s, 72°C for 35 s, followed by melt curve analysis using primers for mouse mtDNA encompassing D-loop; the binding site of POLG: mPOLG ChIP F, TGA TCA ATT CTA GTA GTT CCC AAA A; mPOLG ChIP R, ACC TCT AAT TAA TTA TAA GGC CAGG. ..

Article Title: In cell mutational interference mapping experiment (in cell MIME) identifies the 5′ polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging
Article Snippet: .. Samples were then run on a 1% agarose gel and the range corresponding to 200–600 bp range was isolated, and purified using Nucleospin Gel and PCR Clean-up columns. .. DNA libraries were quantified by qPCR using Illumina PE PCR primer 1.0 and one of the Illumina Index primers (for multiplexing) with Brilliant II SYBR master mix.

Polymerase Chain Reaction:

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells.
Article Snippet: .. After extensive washing, chromatin was eluted and treated with RNAseA and proteinase K to reverse the crosslinking, followed by purification using PCR clean-up columns (MachereyNagel). qRT-PCRwas then run using the Eva Green system (Solis Biodyne) at 95 C for 12min, 40 cycles of 95 C for 15 s, 60 C for 20 s, 72 C for 35 s, followed by melt curve analysis using primers for mouse mtDNA encompassing D-loop; the binding site of POLG: mPOLG ChIP F, TGA TCA ATT CTA GTA GTT CCC AAA A; mPOLG ChIP R, ACC TCT AAT TAA TTA TAA GGC CAGG. ..

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

Article Title: NG2 expression in NG2 glia is regulated by binding of SoxE and bHLH transcription factors to a Cspg4 intronic enhancer
Article Snippet: .. Cross-links were reversed by incubating the eluted material in proteinase K at 65o C for 2 hours, and DNA was purified using NucleoSpin Gel and PCR Clean-up columns (Macherey-Nagel, USA). .. The purified DNA was subjected to PCR using int1–3b and intron 2 primers from the mouse Cspg4 gene ( ) and the products were separated on 1.8% agarose gels.

Article Title: Introns Regulate Gene Expression in Cryptococcus neoformans in a Pab2p Dependent Pathway
Article Snippet: .. The cDNA was column-purified using NucleoSpin Gel and PCR Clean-up columns (MACHEREY-NAGEL). .. Specific PCR products were analysed by 2% high resolution agarose gel (Ultra pure 1000; Life Technologies) pre-stained with sybr safe (Life Technologies) and imaged against a 100 bp ladder (New England Biolabs) using an LAS 3000 imager and multigauge software (Fujifilm).

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells
Article Snippet: .. After extensive washing, chromatin was eluted and treated with RNAseA and proteinase K to reverse the crosslinking, followed by purification using PCR clean-up columns (MachereyNagel). qRT-PCR was then run using the Eva Green system (Solis Biodyne) at 95°C for 12 min, 40 cycles of 95°C for 15 s, 60°C for 20 s, 72°C for 35 s, followed by melt curve analysis using primers for mouse mtDNA encompassing D-loop; the binding site of POLG: mPOLG ChIP F, TGA TCA ATT CTA GTA GTT CCC AAA A; mPOLG ChIP R, ACC TCT AAT TAA TTA TAA GGC CAGG. ..

Article Title: In cell mutational interference mapping experiment (in cell MIME) identifies the 5′ polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging
Article Snippet: .. Samples were then run on a 1% agarose gel and the range corresponding to 200–600 bp range was isolated, and purified using Nucleospin Gel and PCR Clean-up columns. .. DNA libraries were quantified by qPCR using Illumina PE PCR primer 1.0 and one of the Illumina Index primers (for multiplexing) with Brilliant II SYBR master mix.

Sequencing:

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

Binding Assay:

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells.
Article Snippet: .. After extensive washing, chromatin was eluted and treated with RNAseA and proteinase K to reverse the crosslinking, followed by purification using PCR clean-up columns (MachereyNagel). qRT-PCRwas then run using the Eva Green system (Solis Biodyne) at 95 C for 12min, 40 cycles of 95 C for 15 s, 60 C for 20 s, 72 C for 35 s, followed by melt curve analysis using primers for mouse mtDNA encompassing D-loop; the binding site of POLG: mPOLG ChIP F, TGA TCA ATT CTA GTA GTT CCC AAA A; mPOLG ChIP R, ACC TCT AAT TAA TTA TAA GGC CAGG. ..

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells
Article Snippet: .. After extensive washing, chromatin was eluted and treated with RNAseA and proteinase K to reverse the crosslinking, followed by purification using PCR clean-up columns (MachereyNagel). qRT-PCR was then run using the Eva Green system (Solis Biodyne) at 95°C for 12 min, 40 cycles of 95°C for 15 s, 60°C for 20 s, 72°C for 35 s, followed by melt curve analysis using primers for mouse mtDNA encompassing D-loop; the binding site of POLG: mPOLG ChIP F, TGA TCA ATT CTA GTA GTT CCC AAA A; mPOLG ChIP R, ACC TCT AAT TAA TTA TAA GGC CAGG. ..

Chromatin Immunoprecipitation:

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells.
Article Snippet: .. After extensive washing, chromatin was eluted and treated with RNAseA and proteinase K to reverse the crosslinking, followed by purification using PCR clean-up columns (MachereyNagel). qRT-PCRwas then run using the Eva Green system (Solis Biodyne) at 95 C for 12min, 40 cycles of 95 C for 15 s, 60 C for 20 s, 72 C for 35 s, followed by melt curve analysis using primers for mouse mtDNA encompassing D-loop; the binding site of POLG: mPOLG ChIP F, TGA TCA ATT CTA GTA GTT CCC AAA A; mPOLG ChIP R, ACC TCT AAT TAA TTA TAA GGC CAGG. ..

Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells
Article Snippet: .. After extensive washing, chromatin was eluted and treated with RNAseA and proteinase K to reverse the crosslinking, followed by purification using PCR clean-up columns (MachereyNagel). qRT-PCR was then run using the Eva Green system (Solis Biodyne) at 95°C for 12 min, 40 cycles of 95°C for 15 s, 60°C for 20 s, 72°C for 35 s, followed by melt curve analysis using primers for mouse mtDNA encompassing D-loop; the binding site of POLG: mPOLG ChIP F, TGA TCA ATT CTA GTA GTT CCC AAA A; mPOLG ChIP R, ACC TCT AAT TAA TTA TAA GGC CAGG. ..

Plasmid Preparation:

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish
Article Snippet: .. To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers. ..

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    MACHEREY NAGEL pcr clean up spin columns
    Real-time <t>PCR</t> analysis shows loss of endonuclease expression and associated loss of editing. Relative RNA abundance is shown for KREN1, KREN2, KREN3 and never-edited mRNAs COI and ND4 (black bars), pre-edited mRNAs (white bars), and edited mRNAs (gray bars). For each target <t>amplicon,</t> the relative change in RNA abundance was determined by using telomerase reverse transcriptase (TERT) mRNA as an internal control, with each cell line compared relative to either 427wt or MGA control as indicated. ( A ) MGA cells have no large changes in mRNA abundance relative to 427wt, with the exception of the complete loss of CYb editing. As the remaining cell lines are compared to MGA, CYb is excluded from those analyses. ( B ) KREN1 only cells have no detectable KREN2 or KREN3 mRNA, and a broad loss of RNA editing. ( C ) KREN2 only cells have no detectable KREN1 or KREN3 mRNA, and a broad loss of RNA editing. ( D ) KREN3 only cells have no detectable KREN1 or KREN2 mRNA, and a broad loss of RNA editing, with COII editing notably retained. ( E ) Triple null cells have no detectable KREN1, KREN2, or KREN3 mRNA, and a broad loss of RNA editing. ( F ) KREN2 null cells have no detectable KREN2 mRNA, and a broad loss of RNA editing with some amount of COII and ND7 editing retained. ( G ) KREN3 null cells have no detectable KREN3 mRNA, and a loss of COII RNA editing with variable amounts of other edited mRNAs retained.
    Pcr Clean Up Spin Columns, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr clean up spin columns/product/MACHEREY NAGEL
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    pcr clean up spin columns - by Bioz Stars, 2020-09
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    89
    MACHEREY NAGEL pcr clean up columns
    Characterization of a stable <t>eys</t> rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of <t>RT-PCR</t> analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.
    Pcr Clean Up Columns, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 89/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr clean up columns/product/MACHEREY NAGEL
    Average 89 stars, based on 33 article reviews
    Price from $9.99 to $1999.99
    pcr clean up columns - by Bioz Stars, 2020-09
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    Image Search Results


    Real-time PCR analysis shows loss of endonuclease expression and associated loss of editing. Relative RNA abundance is shown for KREN1, KREN2, KREN3 and never-edited mRNAs COI and ND4 (black bars), pre-edited mRNAs (white bars), and edited mRNAs (gray bars). For each target amplicon, the relative change in RNA abundance was determined by using telomerase reverse transcriptase (TERT) mRNA as an internal control, with each cell line compared relative to either 427wt or MGA control as indicated. ( A ) MGA cells have no large changes in mRNA abundance relative to 427wt, with the exception of the complete loss of CYb editing. As the remaining cell lines are compared to MGA, CYb is excluded from those analyses. ( B ) KREN1 only cells have no detectable KREN2 or KREN3 mRNA, and a broad loss of RNA editing. ( C ) KREN2 only cells have no detectable KREN1 or KREN3 mRNA, and a broad loss of RNA editing. ( D ) KREN3 only cells have no detectable KREN1 or KREN2 mRNA, and a broad loss of RNA editing, with COII editing notably retained. ( E ) Triple null cells have no detectable KREN1, KREN2, or KREN3 mRNA, and a broad loss of RNA editing. ( F ) KREN2 null cells have no detectable KREN2 mRNA, and a broad loss of RNA editing with some amount of COII and ND7 editing retained. ( G ) KREN3 null cells have no detectable KREN3 mRNA, and a loss of COII RNA editing with variable amounts of other edited mRNAs retained.

    Journal: Nucleic Acids Research

    Article Title: In vivo cleavage specificity of Trypanosoma brucei editosome endonucleases

    doi: 10.1093/nar/gkx116

    Figure Lengend Snippet: Real-time PCR analysis shows loss of endonuclease expression and associated loss of editing. Relative RNA abundance is shown for KREN1, KREN2, KREN3 and never-edited mRNAs COI and ND4 (black bars), pre-edited mRNAs (white bars), and edited mRNAs (gray bars). For each target amplicon, the relative change in RNA abundance was determined by using telomerase reverse transcriptase (TERT) mRNA as an internal control, with each cell line compared relative to either 427wt or MGA control as indicated. ( A ) MGA cells have no large changes in mRNA abundance relative to 427wt, with the exception of the complete loss of CYb editing. As the remaining cell lines are compared to MGA, CYb is excluded from those analyses. ( B ) KREN1 only cells have no detectable KREN2 or KREN3 mRNA, and a broad loss of RNA editing. ( C ) KREN2 only cells have no detectable KREN1 or KREN3 mRNA, and a broad loss of RNA editing. ( D ) KREN3 only cells have no detectable KREN1 or KREN2 mRNA, and a broad loss of RNA editing, with COII editing notably retained. ( E ) Triple null cells have no detectable KREN1, KREN2, or KREN3 mRNA, and a broad loss of RNA editing. ( F ) KREN2 null cells have no detectable KREN2 mRNA, and a broad loss of RNA editing with some amount of COII and ND7 editing retained. ( G ) KREN3 null cells have no detectable KREN3 mRNA, and a loss of COII RNA editing with variable amounts of other edited mRNAs retained.

    Article Snippet: PCR amplicon DNA was isolated from gel slices using NuceloSpin Gel and PCR Clean-up spin columns, eluting in 30 μl volume (Macherey-Nagel).

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

    Characterization of a stable eys rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.

    Journal: PLoS ONE

    Article Title: Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish

    doi: 10.1371/journal.pone.0200789

    Figure Lengend Snippet: Characterization of a stable eys rmc101/rmc101 zebrafish line. (A) Sanger sequencing identified a five base pair deletion in exon 20 in eys rmc101/rmc101 zebrafish. (B) Representative gel image of RT-PCR analysis using RNA from a pool of larvae (n = 15), which shows that eys transcripts are present in both wild-type and eys rmc101/rmc101 zebrafish (upper panel). Sanger sequencing confirmed the presence of the five base pair deletion in the eys rmc101/rmc101 transcript (lower panel). (C) Protein domain structures of wild-type Eys and the truncated Eys protein that is predicted in eys rmc101/rmc101 zebrafish.

    Article Snippet: To verify that the amplified products indeed correspond to eys , PCR products were purified on Nucleospin Gel and PCR Clean-up columns (Macherey Nagel) and either directly sequenced or cloned in the pCR4-TOPO vector with the use of the TOPO TA Cloning Kit (Invitrogen) for sequencing with T7 and T3 sequencing primers.

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction

    mtDNA Is Replenished and Respiration Recovers Early in Tumor Formation by 4T1ρ° Cells (A) BALB/c mice (n = 6) were grafted subcutaneously (s.c.) with 4T1 or 4T1 ρ° cells at 10 6 per animal, and tumor volume was assessed by ultrasound imaging (USI) (n = 6). (B and C) Time schedule of retrieval of pre-tumor plaques and tumors from BALB/c mice is shown in (B), as indicated in (C) for D5 tissue. (D) Individual lines retrieved from mice according to the schedule in (B) were assessed for uncoupled (ETC), routine, and leak respiration using the Oxygraph (n = 3). (E) Cell lines were evaluated for the presence of mtDNA using probes against a polymorphism in either 4T1 or host 16S rRNA by sc/qPCR; 92 cells per line were assessed. (F) Distribution of mtDNA polymorphism in 16S rRNA of individual cells of lines plotted using data presented in (E). (G) mTRIP assay in the presence of proteinase K was used to detect the mtDNA initiation of replication marker (mREP) and global mitochondrial transcripts (mTRANS) unmasked from proteins, in single cells (n = 3; 100 cells were assessed per condition). (H) Cell lines were assessed for binding of TFAM and POLG1 to the D LOOP region of mtDNA using a mitoChIP assay (n = 3). (I) Individual lines were probed for the level of mtDNA-processing proteins using WB. (J–L) qRT-PCR was used to assess the level of transcripts of selected subunits of mitochondrially encoded transcripts of RCs (n = 3) (J). Selected subunits of mitochondrial RCs were evaluated using WB in individual lines (K), which were then assessed by NBGE for the assembly of RCs and SCs using antibodies to relevant subunits, as shown (L). Data in (A), (D), (G), (H), and (J) are mean values ± SD. Representative images of three biological replicates are shown for (I), (K), and (L).

    Journal: Cell metabolism

    Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells

    doi: 10.1016/j.cmet.2018.10.014

    Figure Lengend Snippet: mtDNA Is Replenished and Respiration Recovers Early in Tumor Formation by 4T1ρ° Cells (A) BALB/c mice (n = 6) were grafted subcutaneously (s.c.) with 4T1 or 4T1 ρ° cells at 10 6 per animal, and tumor volume was assessed by ultrasound imaging (USI) (n = 6). (B and C) Time schedule of retrieval of pre-tumor plaques and tumors from BALB/c mice is shown in (B), as indicated in (C) for D5 tissue. (D) Individual lines retrieved from mice according to the schedule in (B) were assessed for uncoupled (ETC), routine, and leak respiration using the Oxygraph (n = 3). (E) Cell lines were evaluated for the presence of mtDNA using probes against a polymorphism in either 4T1 or host 16S rRNA by sc/qPCR; 92 cells per line were assessed. (F) Distribution of mtDNA polymorphism in 16S rRNA of individual cells of lines plotted using data presented in (E). (G) mTRIP assay in the presence of proteinase K was used to detect the mtDNA initiation of replication marker (mREP) and global mitochondrial transcripts (mTRANS) unmasked from proteins, in single cells (n = 3; 100 cells were assessed per condition). (H) Cell lines were assessed for binding of TFAM and POLG1 to the D LOOP region of mtDNA using a mitoChIP assay (n = 3). (I) Individual lines were probed for the level of mtDNA-processing proteins using WB. (J–L) qRT-PCR was used to assess the level of transcripts of selected subunits of mitochondrially encoded transcripts of RCs (n = 3) (J). Selected subunits of mitochondrial RCs were evaluated using WB in individual lines (K), which were then assessed by NBGE for the assembly of RCs and SCs using antibodies to relevant subunits, as shown (L). Data in (A), (D), (G), (H), and (J) are mean values ± SD. Representative images of three biological replicates are shown for (I), (K), and (L).

    Article Snippet: After extensive washing, chromatin was eluted and treated with RNAseA and proteinase K to reverse the crosslinking, followed by purification using PCR clean-up columns (MachereyNagel). qRT-PCR was then run using the Eva Green system (Solis Biodyne) at 95°C for 12 min, 40 cycles of 95°C for 15 s, 60°C for 20 s, 72°C for 35 s, followed by melt curve analysis using primers for mouse mtDNA encompassing D-loop; the binding site of POLG: mPOLG ChIP F, TGA TCA ATT CTA GTA GTT CCC AAA A; mPOLG ChIP R, ACC TCT AAT TAA TTA TAA GGC CAGG.

    Techniques: Mouse Assay, Imaging, Real-time Polymerase Chain Reaction, Marker, Binding Assay, Western Blot, Quantitative RT-PCR

    Respiration Recovery Is Associated with Reactivation of DHODH (A) Cell lines as shown were evaluated for growth in the presence of uridine and pyruvate, or with pyruvate or uridine removed. (B) Cell lines were assessed for DHODH expression by qRT-PCR and WB. (C–E) DHODH-dependent respiration was assessed in parental 4T1 cells in the absence and presence of 30 μM leflunomide (Lef) (C). Individual lines were assessed for DHODH-dependent respiration (D) and for the orotate-to-DHO ratio (E). (F) Parental, D0, D5, D10, D15, D20, D25, and D60 cells (10 6 ) were grafted s.c. into BALB/c mice, and tumor growth was evaluated by USI. (G) Tumors grown from parental or 4T1 ρ° cells were excised and processed in a dedicated tissue shredder, and the homogenates were assessed for CI-, CII-, and DHODH-dependent respiration using an Oxygraph. (H) BALB/c mice were grafted s.c. with 10 6 D0 cells. On the days indicated, animals were sacrificed and tissue from the grafted region excised, sectioned, and assessed by immunohistochemistry for proliferation using anti-Ki67 IgG followed by confocal microscopy. The numbers indicate percentage of Ki67-positive cells. Data in (A) are mean values derived from two biological replicates with differences less than 10%; data in (B) are mean values ±SD (n ≥ 3); data in (C–G) are mean values ± SEM (n = 3); images in (B) and (H) are representative of three independent experiments. The symbol “*” indicates statistically significant differences from parental cells, and the symbol “#” indicates statistically significant difference from D0 cells, with p

    Journal: Cell metabolism

    Article Title: Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells

    doi: 10.1016/j.cmet.2018.10.014

    Figure Lengend Snippet: Respiration Recovery Is Associated with Reactivation of DHODH (A) Cell lines as shown were evaluated for growth in the presence of uridine and pyruvate, or with pyruvate or uridine removed. (B) Cell lines were assessed for DHODH expression by qRT-PCR and WB. (C–E) DHODH-dependent respiration was assessed in parental 4T1 cells in the absence and presence of 30 μM leflunomide (Lef) (C). Individual lines were assessed for DHODH-dependent respiration (D) and for the orotate-to-DHO ratio (E). (F) Parental, D0, D5, D10, D15, D20, D25, and D60 cells (10 6 ) were grafted s.c. into BALB/c mice, and tumor growth was evaluated by USI. (G) Tumors grown from parental or 4T1 ρ° cells were excised and processed in a dedicated tissue shredder, and the homogenates were assessed for CI-, CII-, and DHODH-dependent respiration using an Oxygraph. (H) BALB/c mice were grafted s.c. with 10 6 D0 cells. On the days indicated, animals were sacrificed and tissue from the grafted region excised, sectioned, and assessed by immunohistochemistry for proliferation using anti-Ki67 IgG followed by confocal microscopy. The numbers indicate percentage of Ki67-positive cells. Data in (A) are mean values derived from two biological replicates with differences less than 10%; data in (B) are mean values ±SD (n ≥ 3); data in (C–G) are mean values ± SEM (n = 3); images in (B) and (H) are representative of three independent experiments. The symbol “*” indicates statistically significant differences from parental cells, and the symbol “#” indicates statistically significant difference from D0 cells, with p

    Article Snippet: After extensive washing, chromatin was eluted and treated with RNAseA and proteinase K to reverse the crosslinking, followed by purification using PCR clean-up columns (MachereyNagel). qRT-PCR was then run using the Eva Green system (Solis Biodyne) at 95°C for 12 min, 40 cycles of 95°C for 15 s, 60°C for 20 s, 72°C for 35 s, followed by melt curve analysis using primers for mouse mtDNA encompassing D-loop; the binding site of POLG: mPOLG ChIP F, TGA TCA ATT CTA GTA GTT CCC AAA A; mPOLG ChIP R, ACC TCT AAT TAA TTA TAA GGC CAGG.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Mouse Assay, Immunohistochemistry, Confocal Microscopy, Derivative Assay

    ChIP to show binding of Olig2 and Sox10 and active histone marks at int1–3b. (A) Scheme of the Cspg4 gene showing the location of int1–3b and intron2 primers at the top. Arrows indicate primers used for ChIP-PCR. The image of a gel below shows PCR detection of int1–3b or intron 2 regions on immunoprecipitates formed from Oli-neu cell nuclear extracts using control rabbit IgG, rabbit anti-histone H3 antibody (positive control), rabbit anti-Olig2 antibody, and rabbit anti-Sox10 antibody. The expected size of int1–3b PCR product is 126 bp and is indicated by an arrowhead on the left margin. The expected size of intron 2 PCR product is 136 bp and is indicated by an arrowhead on the right margin. The positions of 200 bp and 100 bp on the DNA ladder are indicated by arrows. The faster migrating band across all lanes at the bottom indicated by an asterisk are likely to be primer dimers. (B) Genome browser tracks at the int1–3b and int2 loci showing ChIP-seq tracks from epiblast stem cell-derived OPCs. H3K4me1 and H3K27ac enhancer-associated histone modifications are enriched at int1–3b, but not at int2.

    Journal: Glia

    Article Title: NG2 expression in NG2 glia is regulated by binding of SoxE and bHLH transcription factors to a Cspg4 intronic enhancer

    doi: 10.1002/glia.23521

    Figure Lengend Snippet: ChIP to show binding of Olig2 and Sox10 and active histone marks at int1–3b. (A) Scheme of the Cspg4 gene showing the location of int1–3b and intron2 primers at the top. Arrows indicate primers used for ChIP-PCR. The image of a gel below shows PCR detection of int1–3b or intron 2 regions on immunoprecipitates formed from Oli-neu cell nuclear extracts using control rabbit IgG, rabbit anti-histone H3 antibody (positive control), rabbit anti-Olig2 antibody, and rabbit anti-Sox10 antibody. The expected size of int1–3b PCR product is 126 bp and is indicated by an arrowhead on the left margin. The expected size of intron 2 PCR product is 136 bp and is indicated by an arrowhead on the right margin. The positions of 200 bp and 100 bp on the DNA ladder are indicated by arrows. The faster migrating band across all lanes at the bottom indicated by an asterisk are likely to be primer dimers. (B) Genome browser tracks at the int1–3b and int2 loci showing ChIP-seq tracks from epiblast stem cell-derived OPCs. H3K4me1 and H3K27ac enhancer-associated histone modifications are enriched at int1–3b, but not at int2.

    Article Snippet: Cross-links were reversed by incubating the eluted material in proteinase K at 65o C for 2 hours, and DNA was purified using NucleoSpin Gel and PCR Clean-up columns (Macherey-Nagel, USA).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction, Positive Control, Derivative Assay