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: PLoS ONE

Article Title: A MiRNA Signature for Defining Aggressive Phenotype and Prognosis in Gliomas

doi: 10.1371/journal.pone.0108950

Figure Lengend Snippet: Validation of microarray data by qRT PCR analysis. The 32 glioma samples and 4 NBT were analyzed by RT-qPCR for the expression of the 22 miRNAs identified by microarray profiling. Boxplots of miRNAs expression in normal brain tissues (NBT), WHO grade II gliomas (II) and WHO grade III and IV gliomas (III+IV). Expression levels are expressed as: target miRNA/RNU48 multiplied by 1000. The boxes mark the interquartile range, (interval between the 25th and 75th percentile). The lines inside the boxes denote median values, the symbol * denote the outliers. #NBT vs II vs III+IV comparison by Kruskall Wallis Test; §II vs III+IV comparison by Mann Whitney test.

Article Snippet: PCR fragments for each of the 22 miRNAs and RNU48 endogenous control were generated using TaqMan miRNA assay, cloned in the StrataClone PCR Cloning Vector pSC-A (Stratagene, Santa Clara, CA) and introduced in StrataClone SoloPack Competent Cells.

Techniques: Microarray, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Hunting the Extinct Steppe Bison (Bison priscus) Mitochondrial Genome in the Trois-Frères Paleolithic Painted Cave

doi: 10.1371/journal.pone.0128267

Figure Lengend Snippet: Characteristics of the sequences used to assemble the Bison priscus mitochondrial genome. (A) Size distribution of the 3,851 unique Illumina reads. (B) Coverage for each position of the SGE2seq sequence obtained by merging the sequences of Illumina reads and PCR fragments.

Article Snippet: The final sequence, termed SGE2seq, was established using 81 consensus sequences of PCR fragments to complement the information derived from the Illumina reads.

Techniques: Sequencing, Polymerase Chain Reaction

Journal: Viruses

Article Title: A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus

doi: 10.3390/v11121129

Figure Lengend Snippet: Host and viral DNA content of differently treated ASFV samples. Quantitative dual PCR was executed by Virotype ASFV PCR Kit. C t values of individual and averaged samples represented by colored circles and grey box respectively. Averages were calculated from numbers of samples indicated on X axis. Gray circles indicate minimum and maximum values. C t values higher than 40 (undetectable host DNA) are represented by 41. Whole genome amplification (WGA) was executed by REPLI-g Mini Kit.

Article Snippet: The barcoded library DNA samples were purified using the Gel/PCR DNA fragments extraction kit (Geneaid Biotech, Ltd., Taipei, Taiwan) according to the manufacturer’s instructions.

Techniques: Polymerase Chain Reaction, Whole Genome Amplification

Journal: BMC Genomics

Article Title: The MalR type regulator AcrC is a transcriptional repressor of acarbose biosynthetic genes in Actinoplanes sp. SE50/110

doi: 10.1186/s12864-017-3941-x

Figure Lengend Snippet: Electrophoretic mobility shift assays with AcrC protein and the intergenic region of acbE and acbD . a EMSAs with the 342 bp fragment of the intergenic region of acbE / acbD , the 217 bp intergenic region malE / acrC as well as the 203 bp region dapE/ACSP50_6389 . 0.05 pmol Cy3 labeled PCR fragments were incubated with 80 pmol purified AcrC protein, 0.05 μg herring sperm DNA for blocking of unspecific binding, and 100 mg BSA. 12.5 pmol unlabeled double-stranded oligonucleotides (ds oligo) covering the acrC site plus 5 bp up- and downstream were added as indicated. Separation was carried out with 10% native polyacrylamide (TBE) gels and visualized by fluorescence imaging. b Intergenic region of acbE and acbD used for the EMSAs with the promoter motives described in [ 14 ] and the acrC binding sites. c Intergenic region of malE and acrC used for the EMSA with promoter motives

Article Snippet: Cy3-labeled primers (Metabion, Steinkirchen, Germany) were used to produce PCR fragments, which were then purified by using a PCR Clean Up Kit (Macherey Nagel, Düren, Germany).

Techniques: Electrophoretic Mobility Shift Assay, Labeling, Polymerase Chain Reaction, Incubation, Purification, Blocking Assay, Binding Assay, Fluorescence, Imaging

Journal: Scientific Reports

Article Title: A semi-automated luminescence based standard membrane feeding assay identifies novel small molecules that inhibit transmission of malaria parasites by mosquitoes

doi: 10.1038/srep18704

Figure Lengend Snippet: ( A ) Schematic of the gene targeting strategy to insert a GFP-Luc reporter gene under control of the hsp70 promoter in the pfs47 locus, showing the genomic locus of the wild-type (WT) and gene deletion mutants ∆pf47 containing the GFP-luciferase ORF before and after removal of the hdhfr resistance marker. P1, P2 : primers MWV300 and MWV301, respectively, used for PCR analysis; X ( XcmI ), N ( NcoI ): restriction sites used for conformation of LR-PCR analysis; pf47 : pf47 locus; hrp: histidine rich protein; cam: calmodulin; hsp: heat shock protein; hdhfr : human dihydrodrofolate reductase coding region; GFP-Luc : GFP-luciferase fusion protein; fcu: cytosine deaminase/uracil phosphoribosyl transferase; pbdt: P. berghei dhfr transcription termination region; kb: kilo basepairs. ( B ) Verification of the integration site by PCR and restriction digestion. Genomic DNA fragments from the parental NF54 strain (WT), intermediate transgene NF54-HGL-hDHFR and final transgene NF54-HGL were amplified by PCR using primers MWV300 and MWV301 (P1 and P2) and analysed by DNA gel electrophoresis. Where indicated, the PCR fragment was subjected to restriction digestion with XcmI/NcoI (X/N) ( C ) Luminescence intensity (relative light units, RLU) as a function of cell number. The figure shows a comparison between uninfected red blood cells (RBC), immature gametocytes (Day 8 GCT), mature gametocytes (Day 14 GCT) and asexual blood stage parasites. ( D–G ) FLP-FRT mediated excision of the selection marker restores sensitivity to antifolates. Drug sensitivity analysis of WT, ∆Pf47GFP-luc-hdhfr and ∆Pf47GFP-luc to DHA ( D ), Chloroquine ( E ), Pyrimethamine ( F ), and WR99210 ( G ) in a dose dependent asexual growth inhibition assay using pLDH enzyme activity as a readout. Depicted data represents a triplicate measurement and was normalized to the MIN (10 μM DHA) and MAX (0.1% DMSO) controls. Error bars indicate standard deviations.

Article Snippet: Pfs47 target regions, 5′hsp70 and 3′hsp86 fragments were amplified by PCR amplification (Phusion, Finnzymes) from genomic P. falciparum DNA (NF54 strain) and all PCR fragments were sequenced after TOPO TA (Invitrogen) sub-cloning.

Techniques: Luciferase, Marker, Polymerase Chain Reaction, Chick Chorioallantoic Membrane Assay, Amplification, DNA Gel Electrophoresis, Selection, Growth Inhibition Assay, Activity Assay

Journal: PLoS Pathogens

Article Title: IFN-Lambda (IFN-?) Is Expressed in a Tissue-Dependent Fashion and Primarily Acts on Epithelial Cells In Vivo

doi: 10.1371/journal.ppat.1000017

Figure Lengend Snippet: Quantification of IFN-λ, IFN-α5, and IFN-β transcripts in virus-infected brains and livers. Histograms show the number of IFN cDNA copies per 10 6 β-actin cDNA copies, determined by real-time PCR, after reverse transcription of RNA extracted from the brain and liver of mice infected with different RNA viruses, in different experimental settings (see Table 2 ). A, B, F, G, H: mean and standard deviation of groups of mice. C–E: data from individual mice. Background amplification in mock-infected mice (not shown) was less than 1 copy of IFN-β or IFN-λ, and less than 10 copies of IFN-α5 cDNA, per 10 6 copies of β-actin cDNA.

Article Snippet: Standards consisted of 10-fold dilutions of known concentrations of murine genomic DNA, of plasmids carrying the PCR fragment of interest (pCR4-Topo, Invitrogen) or plasmid pcDNA3-IFN-α5 or pEF-IFN-λ3 (kindly provided by S. Kotenko).

Techniques: Infection, Real-time Polymerase Chain Reaction, Mouse Assay, Standard Deviation, Amplification

Journal: PLoS Pathogens

Article Title: IFN-Lambda (IFN-?) Is Expressed in a Tissue-Dependent Fashion and Primarily Acts on Epithelial Cells In Vivo

doi: 10.1371/journal.ppat.1000017

Figure Lengend Snippet: OASl2 expression in different tissues after electroinjection of plasmids coding for IFN-α or IFN-λ. OASl2 transcripts detected by real-time RT-PCR, 7 days after electroinjection of plasmid coding for MuIFN-α6T, MuIFN-α6T/D78N, MuIFN-λ3 or the empty vector (mock), in 7 week-old FVB/N mice. Results are expressed as OASl2 cDNA copies per β-actin cDNA copy. Graphs present results for individual mice and the mean for each group, for one representative experiment.

Article Snippet: Standards consisted of 10-fold dilutions of known concentrations of murine genomic DNA, of plasmids carrying the PCR fragment of interest (pCR4-Topo, Invitrogen) or plasmid pcDNA3-IFN-α5 or pEF-IFN-λ3 (kindly provided by S. Kotenko).

Techniques: Expressing, Quantitative RT-PCR, Plasmid Preparation, Mouse Assay

Journal: PLoS Pathogens

Article Title: IFN-Lambda (IFN-?) Is Expressed in a Tissue-Dependent Fashion and Primarily Acts on Epithelial Cells In Vivo

doi: 10.1371/journal.ppat.1000017

Figure Lengend Snippet: Relative expression of the various IFN-α subtypes in MHV-A59 infected livers. IFN-α coding sequences were amplified by RT-PCR using a primer mixture designed to amplify equally the different murine IFN-α subtypes [33] . PCR products were then cloned and individual clones were sequenced. The histogram shows the percentage of sequences from 2 mice (50 and 53 sequences) corresponding to each IFN-α subtype.

Article Snippet: Standards consisted of 10-fold dilutions of known concentrations of murine genomic DNA, of plasmids carrying the PCR fragment of interest (pCR4-Topo, Invitrogen) or plasmid pcDNA3-IFN-α5 or pEF-IFN-λ3 (kindly provided by S. Kotenko).

Techniques: Expressing, Infection, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Clone Assay, Mouse Assay

Journal: PLoS Pathogens

Article Title: IFN-Lambda (IFN-?) Is Expressed in a Tissue-Dependent Fashion and Primarily Acts on Epithelial Cells In Vivo

doi: 10.1371/journal.ppat.1000017

Figure Lengend Snippet: Mx1 gene expression induced by systemic IFN-α and IFN-λ, in organs of IFNAR1-positive and IFNAR1-deficient mice. Mx1 gene transcription was analyzed by real-time RT-PCR, 7 days after electroinjection of plasmid coding for MuIFN-α6T, MuIFN-λ3 or the empty vector (mock) in 6 week-old Mx1-positive mice (2 BALB.A2G-Mx1 and 2 B6.A2G-Mx1 mice, grouped as Mx1/WT mice) or in 8 week-old Mx1/IFNAR1-KO mice. Results are expressed as Mx1 cDNA copies per β-actin cDNA copy. Graphs present results for individual mice and the mean for each group, for one representative experiment.

Article Snippet: Standards consisted of 10-fold dilutions of known concentrations of murine genomic DNA, of plasmids carrying the PCR fragment of interest (pCR4-Topo, Invitrogen) or plasmid pcDNA3-IFN-α5 or pEF-IFN-λ3 (kindly provided by S. Kotenko).

Techniques: Expressing, Mouse Assay, Quantitative RT-PCR, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: IFN-Lambda (IFN-?) Is Expressed in a Tissue-Dependent Fashion and Primarily Acts on Epithelial Cells In Vivo

doi: 10.1371/journal.ppat.1000017

Figure Lengend Snippet: Correlation between the relative responsiveness of organs to IFN-λ and the relative expression of IL-28Rα and IFNAR1. A. Relative functional influence of IFN-λ over IFN-α in various organs. The histogram shows, for each organ, the ratio between OASl2 expression in response to circulating IFN-λ and OASl2 expression in response to circulating IFN-α (mean of two mice), 7 days after electrotransfer of the expression plasmids. B. Relative expression, measured by real-time RT-PCR, of IL-28Rα and IFNAR1 (mean and SD from 5 mice).

Article Snippet: Standards consisted of 10-fold dilutions of known concentrations of murine genomic DNA, of plasmids carrying the PCR fragment of interest (pCR4-Topo, Invitrogen) or plasmid pcDNA3-IFN-α5 or pEF-IFN-λ3 (kindly provided by S. Kotenko).

Techniques: Expressing, Functional Assay, Mouse Assay, Electrotransfer, Quantitative RT-PCR

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: Applied and Environmental Microbiology

Article Title: A Novel Nonantibiotic, lgt-Based Selection System for Stable Maintenance of Expression Vectors in Escherichia coli and Vibrio cholerae

doi: 10.1128/AEM.02143-17

Figure Lengend Snippet: Schematic representation of the construction of Δ lgt strains of E. coli carrying temperature-sensitive plasmid pMT-lgtVc(ts). DNA fragments carrying a deletion of lgt were generated by primerless PCR and cloned into suicide plasmid pMT-ssB. A kanamycin resistance gene flanked by loxP sites was then inserted in place of the deleted lgt gene. The resulting plasmids were used to create deletions in the chromosomes of E. coli by allelic exchange. The kanamycin resistance gene was then removed with Cre recombinase. A temperature-sensitive (ts) plasmid carrying a nonhomologous lgt gene complements the chromosomal deletion, allowing the survival of the strain at 30°C.

Article Snippet: All DNA sequencing of PCR fragments and plasmids was performed by Eurofins Genomics (Ebersberg, Germany).

Techniques: Plasmid Preparation, Generated, Polymerase Chain Reaction, Clone Assay

Journal: Fungal genetics reports

Article Title: A mus-51 RIP allele for transformation of Neurospora crassa

doi: 10.4148/1941-4765.1001

Figure Lengend Snippet: PCR analysis confirms that resistance to cyclosporin A is due to replacement of the csr-1 + allele with csr-1 Δ ∷hph . A) A diagram of the csr-1 + locus (top) relative to the csr-1 Δ ∷hph vector (bottom). Oligonucleotides P583 and P584 amplify a 3719 bp product from the csr-1 locus when it contains the csr-1 + allele and a 3500 bp PCR product from the locus when it contains the csr-1 Δ ∷hph allele. The restriction endonuclease Not I can be used to distinguish between these two PCR products of similar length. While Not I will digest a csr-1 Δ ∷hph PCR product into two fragments of 2473 and 1027 bp, it should not digest a PCR product from csr-1 + . B) Eleven transformants from the csr-1 + gene deletion assay were analyzed by PCR. The four host strains (host), a cyclosporin A-resistant transformant (CART) from each host, and a cyclosporin A-susceptible transformant (CAST) from each host (if available) were analyzed. The csr-1 locus in each strain was PCR amplified with oligonucleotides P583 and P584, digested with Not I, and analyzed by gel electrophoresis. Only CART strains (lanes 2, 5, 8, 11) and the positive control P8-65 (lane 12) produced PCR products with the expected pattern for the csr-1 Δ ∷hph allele. The transformants were not purified by homokaryon isolation, thus csr-1 + and csr-1 Δ ∷hph specific patterns in some lanes is expected ( e.g ., lanes 2, 8, and 11). The 5.0 and 1.5 kb positions, according to the included DNA ladder, are indicated to the left of the gel image. Lane information: kb) DNA ladder; 1) RZS27.4, host; 2) RZS27.4, CART; 3) RZS27.4, CAST; 4) RZS27.6, host; 5) RZS27.6, CART; 6) RZS27.6, CAST; 7) RZS27.10, host; 8) RZS27.10, CART; 9) RZS27.10, CAST; 10) RZS27.18, host; 11) RZS27.18, CART; 12) P8-65, csr-1 Δ ∷hph control; and 13) F2-26, csr-1 + control.

Article Snippet: The PCR product was purified with an IBI scientific Gel/PCR DNA Fragment Extraction Kit and 500 ng were electroporated into conidia as previously described ( ) with selection for hygromycin resistance.

Techniques: Polymerase Chain Reaction, Plasmid Preparation, DNA Deletion Assay, Amplification, Nucleic Acid Electrophoresis, Positive Control, Produced, Purification, Isolation

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: Oncogenesis

Article Title: Identification of strong intron enhancer in the heparanase gene: effect of functional rs4693608 variant on HPSE enhancer activity in hematological and solid malignancies

doi: 10.1038/s41389-018-0060-8

Figure Lengend Snippet: Structure of six DNA constructs for luciferase reporter assay. The HPSE gene fragment of intron 2 was cloned via PCR using PCR II-TOPO vector. The enhancer fragments were digested with Hin dIII and Xho I and ligated into phosphatase-treated pGL4.26 (luc2/minP/Hygro) vector that was digested with the same enzymes. Six DNA constructs were prepared, three in the sense direction and three in the antisense direction. The constructs were named variant (Vr) A, B, and C, according to the linkage disequilibrium (LD) between SNPs and possibility of allele variants. SNP rs4693608 is marked in bold

Article Snippet: The PCR fragments were amplified from genomic DNA (the Wizard® Genomic DNA Purification kit (Promega, Madison, WI), using the forward and reverse primers (Table ).

Techniques: Construct, Luciferase, Reporter Assay, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, Variant Assay