Journal: BMC Developmental Biology

Article Title: Differential requirement for Dab2 in the development of embryonic and extra-embryonic tissues

doi: 10.1186/1471-213X-13-39

Figure Lengend Snippet: Conditional dab2 deletion by Meox2-Cre and Sox2-Cre transgene. Female dab2 (fl/fl) mice were crossed with male dab2 (df/+);Meox2-Cre or dab2 (df/+);Sox2-Cre mice to produce mosaic mutant mice without deletion in the extraembryonic endoderm. Mice of around the age of 3 months were used for these analyses. (A) An example of PCR genotyping assay using tail DNA of the progenies (#1 to 12) generated from the cross between dab2 (fl/fl) and dab2 (df/+);Meox2-Cre. Specific PCR amplifications to identify the locus for cre, fl, df, and wt were performed either separately or in a combined reaction. Examples of Dab2 immunostainings in kidney of a dab2 (df/fl) (B) and a dab2 (df/fl);Meox2-Cre (C) mouse are shown, with corresponding PCR genotyping results shown at the upper right corner. (D) An example of PCR genotyping assay using tail DNA of the progenies (#71 to 94) generated from crosses between dab2 (fl/fl) and dab2 (df/+);Sox2-Cre. (E) DNA gel resolving the genotyping PCR products shows a comparison of the deletion efficiency generated by Sox2-Cre or Meox2-Cre. Corresponding Dab2 stainings show the extent of Dab2 mosaicism in the kidney sections from dab2 (+/df) (F) , dab2 (fl/df);Sox2-Cre (G) , and dab2 (fl/df);Meox2-Cre (H) mice.

Article Snippet: Each PCR fragment generated was amplified using cloned-Pfu DNA polymerase (Stratagene), which has a proofreading activity that results in high-fidelity DNA replication.

Techniques: Mouse Assay, Mutagenesis, Polymerase Chain Reaction, Genotyping Assay, Generated

Journal: BMC Developmental Biology

Article Title: Differential requirement for Dab2 in the development of embryonic and extra-embryonic tissues

doi: 10.1186/1471-213X-13-39

Figure Lengend Snippet: Conditional targeting strategy and screening for homologous recombination of dab2 gene. (A) Schematic illustration of dab2 gene targeting strategy and gene deletion in mutant mice is shown. The targeting construct was made by inserting a neomycin resistance gene (Neo-R) flanked by Frt sites (closed triangles) between exon 4 and 5. LoxP sites (open triangles) were placed flanking exon 3 and 4. Correct homologous recombination of the targeting construct did not alter dab2 gene but allowed expression of Neo-R for selection of mutant ES clones. Following selection and verification, chimeric and then germline mutant mice were made from the mutant ES cells. The Neo-R cassette was excised by crossing with Flp expression mice to generate the floxed allele. The deletion of exon 4 removed a potential alternative translation start site, indicated as “ATG”. (B) Examples of PCR screening assay of neomycin resistant clones (#25-30) following transfection of linearized targeting construct and drug selection. Each clone was amplified separately for wildtype (886 bp) and recombinant allele (1714 bp, as predicted). Here, clone #28 was identified as positive. In the agarose gel: lane 1: Mw markers; lane 2: control mouse DNA; lane 3: KO construct; lane 4: control WT mix, no DNA; 5: control mix for mutant, no DNA; lanes 6 to 17, amplification using DNA template from clone #25 to 30. (C) Examples of targeted allele in selected clones (#1, #28, and #121). Here, clone #1 was found to lack the loxP1 site, whereas clones #28 and #121 contained all components: LoxP1, 1714 bp; LoxP2, 795 bp; Frt1, 635 bp; Frt2, 837 bp; and Neo, 617 bp.

Article Snippet: Each PCR fragment generated was amplified using cloned-Pfu DNA polymerase (Stratagene), which has a proofreading activity that results in high-fidelity DNA replication.

Techniques: Homologous Recombination, Mutagenesis, Mouse Assay, Construct, Expressing, Selection, Clone Assay, Polymerase Chain Reaction, Screening Assay, Transfection, Amplification, Recombinant, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Neuron-specific ELAV/Hu proteins suppress HuR mRNA during neuronal differentiation by alternative polyadenylation

doi: 10.1093/nar/gkr1114

Figure Lengend Snippet: Overexpression of the neuron-specific Hu Family members (HuB, HuC and HuD) affects alternative polyadenylation of HuR mRNA. ( A ) Real time PCR results of transiently transfected HEK293T cells expressing the indicated FLAG tagged constructs demonstrating increased expression of alternatively polyadenylated HuR 6.0-kb mRNA, while maintaining overall HuR levels (Total). GapDH mRNA level was used as a control. Values were calculated as fold change ratios using ΔΔCT values ±SD. The means and SD's are represented from three independent experiments. Asterisks indicates P

Article Snippet: Membranes were probed with PCR fragments corresponding to the HuR neuron-specific UTR, HuR ORF or B-actin ORF, all of which were labeled with [32P]CTP using the Prime-It II Random Primer Labeling Kit (Stratagene).

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Transfection, Expressing, Construct

Journal: Nucleic Acids Research

Article Title: Neuron-specific ELAV/Hu proteins suppress HuR mRNA during neuronal differentiation by alternative polyadenylation

doi: 10.1093/nar/gkr1114

Figure Lengend Snippet: Effect of alternative polyadenylation on stability and translation of HuR mRNA. ( A ) The 6.0 - kb neuron-specific (NS) HuR mRNA has a shorter half-life than the 2.4 - kb ubiquitously expressed mRNA. Control (ctrl) or differentiated (RA) P19 cells were treated with the transcriptional inhibitor Actinomycin D and total RNA collected at the indicated times. HuR mRNA levels were determined via real time PCR using ΔΔ C t calculations and the results plotted as percentage of the initial transcript remaining. Mean half lives were calculated via linear regression and shown ±SD from three independent experiments. ( B ) Representative immunoblot from HEK293T cells transiently transfected with equimolar amounts of constructs consisting of the HuR ORF fused to the indicated portion of the HuR 3′-UTR, showing decreased protein production from the neuron-specific 6.0-kb HuR mRNA isoform as compared to the ubiquitous 2.4-kb form. Topoisomerase (Topo) is shown as a loading control. ( C ) Western blot showing a progressive decrease in endogenous HuR protein expression in terminally differentiated P19 cells following treatment with the mitotic inhibitor AFC to eliminate non-neuronal cells. Semiquantitative PCR was used to determine relative expression of the HuR 6.0-kb mRNA isoform. ( D ) Western blot showing decreased endogenous HuR protein expression in isolated neuronal but not glial cells following P19 differentiation. Tubulin is shown as a loading control.

Article Snippet: Membranes were probed with PCR fragments corresponding to the HuR neuron-specific UTR, HuR ORF or B-actin ORF, all of which were labeled with [32P]CTP using the Prime-It II Random Primer Labeling Kit (Stratagene).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Construct, Western Blot, Expressing, Polymerase Chain Reaction, Isolation

Journal: Nucleic Acids Research

Article Title: Neuron-specific ELAV/Hu proteins suppress HuR mRNA during neuronal differentiation by alternative polyadenylation

doi: 10.1093/nar/gkr1114

Figure Lengend Snippet: The 6.0 - kb HuR mRNA isoform is expressed during retinoic acid induced neuronal differentiation of P19 embryonic carcinoma cells. P19 cells were differentiated in the presence (RA) or absence (Ctrl) of 5 µM RA for 4 days, then replated and allowed to differentiate for 2–6 days. At each time point, total RNA was isolated via Trizol. ( A ) Northern Blot showing increasing expression of the 6.0 - kb mRNA isoform with either a neuron-specific probe (HuR NS from B ) or a probe detecting all HuR mRNA isoforms (HuR ORF from B). β-Actin and ribosomal RNA (18S/28S) were used as loading controls. MW = molecular weight as determined by position of rRNA 18S and 28S bands (B) Semiquantitative PCR to confirm results of Northern blots shown in A. PCR products show consistent expression of the HuR ORF but increased expression of the 6.0 - kb HuR neuron-specific mRNA isoform (HuR NS). Neuron-specific mRNAs HuC and Neuroligin 3 (Neuro3) were used to verify the progression of P19 neuronal differentiation. ( C ) Western blot analysis of Hu protein expression showing induction of neuronal-specific HuB and D during P19 differentiation. GapDH served as a loading control.

Article Snippet: Membranes were probed with PCR fragments corresponding to the HuR neuron-specific UTR, HuR ORF or B-actin ORF, all of which were labeled with [32P]CTP using the Prime-It II Random Primer Labeling Kit (Stratagene).

Techniques: Isolation, Northern Blot, Expressing, Molecular Weight, Polymerase Chain Reaction, Western Blot

Journal: BMC Veterinary Research

Article Title: First isolation and characterization of Brucella microti from wild boar

doi: 10.1186/s12917-015-0456-z

Figure Lengend Snippet: Results of upgraded “Bruce- and Suis-ladder” PCRs of HUN-Bmi-01 strain. left side Bruce-ladder v2.0 PCR with 1682, 1071, 774, 587, 510, 450, 272, and 152 bp amplicons. M: 100 bp DNA marker, S: B. suis , C: B. canis , O: B. ovis , A: B. abortus . right side Suis-ladder PCR with 774 and 197 bp amplicons. M: 100 bp DNA marker, S1: B. suis biovar. 1, S2: B. suis biovar. 2

Article Snippet: The barcoded library DNA samples were column purified using the Gel/PCR DNA Fragments Extraction kit (Geneaid Biotech Ltd., Taipei, Taiwan).

Techniques: Polymerase Chain Reaction, Marker

Journal: Vaccines

Article Title: Characterization of Aggregatibacter actinomycetemcomitans Serotype b Strains with Five Different, Including Two Novel, Leukotoxin Promoter Structures

doi: 10.3390/vaccines8030398

Figure Lengend Snippet: PCR analysis of the leukotoxin promoter region of all A. actinomycetemcomitans ( Aa ) strains compared in the present study, representing five different ltxCABD promoter types. Sizes (bp) of selected bands in the DNA molecular weight marker (MWS) are indicated.

Article Snippet: DNA sequencing of PCR fragments, where feasible, was done at Eurofins MWG Operon (Ebersberg, Germany).

Techniques: Polymerase Chain Reaction, Molecular Weight, Marker

Journal: Vaccines

Article Title: Characterization of Aggregatibacter actinomycetemcomitans Serotype b Strains with Five Different, Including Two Novel, Leukotoxin Promoter Structures

doi: 10.3390/vaccines8030398

Figure Lengend Snippet: Arbitrarily primed (AP)-PCR genotyping of the seven A. actinomycetemcomitans serotype b strains. Distinct banding patterns distinguish AP-PCR types 1, 2 and “other” (AP-PCR types 3‒11, as defined previously [ 13 ]). Sizes (bp) of selected bands in the DNA molecular weight standard (MWS) are indicated.

Article Snippet: DNA sequencing of PCR fragments, where feasible, was done at Eurofins MWG Operon (Ebersberg, Germany).

Techniques: Polymerase Chain Reaction, Molecular Weight

Journal: Vaccines

Article Title: Characterization of Aggregatibacter actinomycetemcomitans Serotype b Strains with Five Different, Including Two Novel, Leukotoxin Promoter Structures

doi: 10.3390/vaccines8030398

Figure Lengend Snippet: Polymerase chain reaction (PCR) analysis of the leukotoxin promoter region of A. actinomycetemcomitans strains 581-18U, 582-18U and 046-19U. As controls, DNA from a strain with a complete, full-length leukotoxin promoter (ref 1) and from a strain with a JP2 genotype promoter (530-bp deletion; ref 2) was amplified. Sizes (bp) of selected bands in the DNA molecular weight standard (MWS) are indicated.

Article Snippet: DNA sequencing of PCR fragments, where feasible, was done at Eurofins MWG Operon (Ebersberg, Germany).

Techniques: Polymerase Chain Reaction, Amplification, Molecular Weight

Journal: Nucleic Acids Research

Article Title: Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event

doi: 10.1093/nar/gkx171

Figure Lengend Snippet: Fe 2+ or Mn 2+ can replace Mg 2+ as a cofactor for Deep Vent (exo-) DNA polymerase. Reaction mixtures with Fe 2+ or Mg 2+ or Mn 2+ or no divalent cation were analyzed for amount of reactants and products at every other PCR cycle for 24 cycles. Reactant consumption and product yields are identical within the uncertainty of this experiment among ( A ) Fe 2+ , ( B ) Mg 2+ or ( C ) Mn 2+ . ( D ) The divalent-minus controls, which lack divalent cations, did not generate product. Neither did the ‘minus template’ negative control reactions (far left lanes of each gel), taken through 24 cycles, generate product.

Article Snippet: The enzyme was heat inactivated at 80°C for 20 min and linearized DNA purified with an IBI Scientific Gel/PCR DNA Fragment Extraction kit.

Techniques: Polymerase Chain Reaction, Negative Control

Journal: EMBO Molecular Medicine

Article Title: IGSF10 mutations dysregulate gonadotropin‐releasing hormone neuronal migration resulting in delayed puberty

doi: 10.15252/emmm.201606250

Figure Lengend Snippet: Flowchart of exome sequencing filtering outcomes Whole exome sequencing was initially performed on DNA extracted from the peripheral blood leukocytes of 111 individuals from the 18 most extensive families from our cohort (76 with DP and 35 controls). The exome sequences were aligned to the UCSC hg19 reference genome. Picard tools and the genome analysis toolkit were used to mark PCR duplicates, realign around indels, recalibrate quality scores, and call variants. Variants were analyzed further and filtered for potential causal variants using filters for quality control, predicted functional annotation, minor allele frequency (MAF), segregation with trait, variants in multiple families, and biological relevance (see Materials and Methods and Appendix Table S1 for further information on filtering criteria). Targeted exome sequencing using a Fluidigm array of 28 candidate genes identified post‐filtering was then performed in a further 42 families from the same cohort (288 individuals, 178 with DP and 110 controls). Variants post‐targeted resequencing were filtered using the same criteria as the whole exome sequencing data. Rare variant burden testing was performed for all genes selected for targeted resequencing, in order to rank candidate genes post‐targeted resequencing. A multiple comparison adjustment was applied to the set of 28 P ‐values post hoc (Benjamini et al , 2001 ). Screening of 100 further cohort controls was via conventional Sanger sequencing. Functional annotation of the variants as described elsewhere in Materials and Methods. DP, delayed puberty. *data unpublished.

Article Snippet: PCR products were separated on an agarose gel, extracted from gel using HiYield Gel/PCR DNA Fragments Extraction Kit (RBC Bioscience, New Taipei City, Taiwan), and sequenced.

Techniques: Sequencing, Polymerase Chain Reaction, Functional Assay, Variant Assay

Journal: Nature Communications

Article Title: Nuclear moonlighting of cytosolic glyceraldehyde-3-phosphate dehydrogenase regulates Arabidopsis response to heat stress

doi: 10.1038/s41467-020-17311-4

Figure Lengend Snippet: Effect of GAPC on NF-YC10 binding to its target promoters. a PCR verification of the precipitated DNA. Chromatin immunoprecipitation (ChIP) was performed using anti-Flag antibody from heat-treated mesophyll protoplasts that were isolated from the GAPC -altered Arabidopsis lines indicated on the top and transfected with 35S:NF-YC10-Flag . Input DNA (ID; see Methods for details) and DNA precipitated with (+) or without (−) the antibody were used for PCR with primers specific to the promoters of genes indicated on the right. Mock, non-transfected; Ubq10 , ubiquitin10. b Quantification of the precipitated DNA. Quantitative PCR was performed with the DNA samples obtained from a . Values are average ± S.D. and shown as % of PCR product amplified from the input DNA. P values indicate significant difference from WT determined by two-tailed student’s t test ( n = 3 independent pools of protoplasts). Black dots represent individual data points. c NF-YC10 protein abundance. After transfection, total proteins were extracted from the protoplasts and immunoblotting was performed with anti-Flag antibody (top). Coomassie blue staining is shown for loading control (bottom).

Article Snippet: The sample was centrifuged at 5000 × g for 3 min and supernatant was incubated with 0.2 M NaCl at 65 °C for 6 h to reverse crosslinking, then incubated with 1 μg proteinase K at 37 °C for 1 h. DNA was purified using Gel/PCR DNA Fragments Extraction Kit (IBI Scientific) according to manufacturer’s instruction.

Techniques: Binding Assay, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Isolation, Transfection, Real-time Polymerase Chain Reaction, Amplification, Two Tailed Test, Staining

Journal: Nature Communications

Article Title: Nuclear moonlighting of cytosolic glyceraldehyde-3-phosphate dehydrogenase regulates Arabidopsis response to heat stress

doi: 10.1038/s41467-020-17311-4

Figure Lengend Snippet: Expression of heat-inducible genes in GAPC -altered Arabidopsis. Total RNA was extracted from 5-day-old seedlings of GAPC1 -OE ( a ), GAPC2 -OE ( b ), and gapc1gapc2 ( c ) treated at 37 °C for 5 h and quantitative RT-PCR was performed with gene-specific primers. Values are average ± S.D. and shown as fold change to WT (dashed line). P values indicate significant difference from WT determined by two-tailed student’s t test ( n = 3 independent groups of > 10 seedlings). Black dots represent individual data points. Hsf, heat shock transcription factor; Hsp, heat shock protein; At1g75860, DNA ligase; At4g36010, pathogenesis-related thaumatin superfamily protein; LFG4, LIFEGUARD4 (Bax inhibitor-1 family protein); EGY3, ETHYLENE-DEPENDENT GRAVITROPISM-DEFICIENT AND YELLOW-GREEN-LIKE3; CLPB3, CASEIN LYTIC PROTEINASE B3; FBS1, F-BOX STRESS INDUCED1; SAP10, STRESS-ASSOCIATED PROTEIN10; DREB2C, DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2C; At1g75960, AMP-dependent synthetase and ligase family protein; ACS7, 1-AMINO-CYCLOPROPANE-1-CARBOXYLATE SYNTHASE7.

Article Snippet: The sample was centrifuged at 5000 × g for 3 min and supernatant was incubated with 0.2 M NaCl at 65 °C for 6 h to reverse crosslinking, then incubated with 1 μg proteinase K at 37 °C for 1 h. DNA was purified using Gel/PCR DNA Fragments Extraction Kit (IBI Scientific) according to manufacturer’s instruction.

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Binding Assay

Journal: Veterinary Research Forum

Article Title: Comparison of cattle BoLA-DRB3 typing by PCR-RFLP, direct sequencing, and high-resolution DNA melting curve analysis

doi: 10.30466/vrf.2018.90444.2189

Figure Lengend Snippet: Seven profiles of BoLA-DRB3.2 identified by PCR-RFLP

Article Snippet: PCR - RFLP analysis .

Techniques: Polymerase Chain Reaction