Journal: Molecular Plant Pathology

Article Title: Deficiency of the melanin biosynthesis genes SCD1 and THR1 affects sclerotial development and vegetative growth, but not pathogenicity, in Sclerotinia sclerotiorum

doi: 10.1111/mpp.12627

Figure Lengend Snippet: Polymerase chain reaction (PCR) confirmation of gene knockout events in three SCD1 and three THR1 transformants. (A) A 1.3‐kb band representing SCD1 and a 3.7‐kb band representing the transforming DNA were amplified from genomic DNA of the wild‐type (WT) and the three SCD1 transformants, respectively; a 0.6‐kb band representing the SCD1 transcript was amplified from cDNA of the WT only; no band was amplified from RNA samples. (B) A 1.1‐kb band representing THR1 and a 3.8‐kb band representing the transforming DNA were amplified from genomic DNA of the WT and the three THR1 transformants, respectively; a 0.8‐kb band representing the THR1 transcript was amplified from cDNA of the WT only. No band was amplified from RNA samples. (C) A 3.3‐kb band representing the hph gene cassette was amplified from all transformants, but not from the WT. (D) A 0.6‐kb band representing ACT1 was amplified from the WT and all transformants.

Article Snippet: Complementary DNA (cDNA) was synthesized with 200 ng of template RNA by an iScript cDNA Synthesis Kit (Bio‐Rad, Hercules, CA, USA) as recommended by the manufacturer.

Techniques: Polymerase Chain Reaction, Gene Knockout, Amplification

Journal: Blood

Article Title: The H3K27me3 demethylase UTX is a gender-specific tumor suppressor in T-cell acute lymphoblastic leukemia

doi: 10.1182/blood-2014-05-577270

Figure Lengend Snippet: UTX mutations in human T-ALL. (A) DNA sequencing chromatogram showing a UTX mutation in the gDNA of a male primary T-ALL patient sample. The mutation is absent in remission material of the same patient. (B) Graphical representation of the localization of genetic lesions in the UTX protein structure. In-frame deletion/insertion mutations are depicted in orange circles and frameshift mutations in blue circles. (C) Genotyping and allelic expression analysis of SNP rs181547731 in gDNA and cDNA derived from female T-ALL lymphoblasts. (D) Graphical representation of the different mutations (dark orange rectangles) and deletions (light orange rectangles) present in a set of T-ALL oncogenes and tumor suppressor genes in 35 primary T-ALL patient samples. The different T-ALL subgroups include TAL-LMO, TLX3, TLX1, HOXA, and patients for whom the subgroup is unknown. The age subgroups include children (age ≤15 years; dark red rectangles) and adults (age > 15 years; light red rectangles). Male and female T-ALL patient samples are presented in dark blue and light blue rectangles, respectively. JmjC, Jumonji C; TPR, tetratricopeptide repeat.

Article Snippet: After RNA quality assessment, complementary DNA (cDNA) synthesis was performed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories).

Techniques: DNA Sequencing, Mutagenesis, Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: Virus-Like Nanoparticle Vaccine Confers Protection against Toxoplasma gondii

doi: 10.1371/journal.pone.0161231

Figure Lengend Snippet: Construction of pFastBac vectors. (A) Toxoplasma gondii IMC gene was PCR-amplified from cDNA synthesized using a Prime Script 1st Strain CDNA Synthesis Kit using total RNA extracted from T . gondii RH. M denotes DNA marker and lane 1 indicates amplified PCR product. (B) Influenza M1 gene was PCR amplified from total RNA extracted from influenza virus (A/PR/8/34). M denotes DNA marker and lane 2 indicates amplified PCR product. (C and D) Toxoplasma gondii IMC gene and influenza M1 gene were cloned in to pFastBac with EcoRI / XhoI and EcoRI / XhoI enzymes, respectively, resulting in T . gondii IMC plasmid (C) and M1 plasmid (D).

Article Snippet: Complementary DNA (cDNA) was synthesized using a Prime Script 1st Strain CDNA Synthesis Kit (Takara, Japan).

Techniques: Polymerase Chain Reaction, Amplification, Synthesized, Marker, Clone Assay, Plasmid Preparation

Journal: Epigenetics & Chromatin

Article Title: Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR

doi: 10.1186/s13072-018-0200-6

Figure Lengend Snippet: Analysis of allelic Commd1 expression and Zrsr1 -DMR methylation in PA and WT MII oocytes. a MII oocytes were prepared from Commd1 PA(B6)/ + (PWK) females (PA) and Commd1 + (B6)/ + (PWK) females (WT). RT-PCR was performed as in Fig. 1 b. Amplified cDNA (250 bp) was digested with NlaIII (CATG). There are two NlaIII restriction sites in the B6 amplicon, but one of these is absent in the PWK amplicon because of a single nucleotide polymorphism (SNP) between the two strains. Adult brain RNA from each of the strains was used as the control. MW: molecular weight marker. b A 278-bp region in Zrsr1 -DMR containing 15 CpG sites (B6) or 14 CpG sites (PWK) was analyzed via bisulfite sequencing. The alleles were discriminated via an SNP (C in B6 and A in PWK) located in the leftmost CpG site in the B6 sequence. Closed and open circles depict methylated and unmethylated CpGs, respectively

Article Snippet: Expression analysis To analyze Commd1 expression in oocyte and adult tissues, cDNA was synthesized with random primers (Takara, #3802) and reverse transcriptase (TOYOBO, TRT-101), and the Commd1 cDNA was amplified by PCR with the primers Comm-F1 (exon 1) and Comm-R1 (exon 2).

Techniques: Expressing, Methylation, Reverse Transcription Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Methylation Sequencing, Sequencing

Journal: Epigenetics & Chromatin

Article Title: Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR

doi: 10.1186/s13072-018-0200-6

Figure Lengend Snippet: Analysis of Commd1 and Zrsr1 expression and Zrsr1 -DMR methylation in adult mice. a Allelic expression of Commd1 was analyzed as in Fig. 2 a in the brain (Br) and liver (Lv) of Commd1 PA(B6)/ + (PWK) (PA) and Commd1 + (B6)/ + (PWK) (WT) adult F1 mice. MW: molecular weight marker. B6 and PWK are the maternal and paternal alleles in the F1 mice, respectively. b Methylation at Zrsr1 -DMR was analyzed as in Fig. 2 b in the brain of adult F1 mice. Shown is the SNP used to discriminate the parental alleles. c Allelic expression of Zrsr1 was analyzed in the F1 adult brain by direct RT-PCR sequencing of the cDNA, which contains two SNP sites between B6 and PWK alleles. d Total Zrsr1 expression was analyzed in triplicate (shown with n) in the adult F1 brain and liver via TaqMan RT-PCR. Total expression is presented relative to the WT expression level in each tissue. The differences in levels of expression between WT and ΔPA F1 mice were statistically significant in the brain and liver (two-sided Student’s t test, * P

Article Snippet: Expression analysis To analyze Commd1 expression in oocyte and adult tissues, cDNA was synthesized with random primers (Takara, #3802) and reverse transcriptase (TOYOBO, TRT-101), and the Commd1 cDNA was amplified by PCR with the primers Comm-F1 (exon 1) and Comm-R1 (exon 2).

Techniques: Expressing, Methylation, Mouse Assay, Molecular Weight, Marker, Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Epigenetics & Chromatin

Article Title: Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR

doi: 10.1186/s13072-018-0200-6

Figure Lengend Snippet: Structure of the Zrsr1 / Commd1 locus and analysis of Commd1 expression and Zrsr1 -DMR methylation in the oocyte. a Zrsr1 , an approximately 2.8-kb intronless gene, and the first two exons of Commd1 are represented by gray and white boxes, respectively. Distances from the Zrsr1 gene to Commd1 exon 1 and exon 2 are indicated above the gene with double-headed arrows. The schematic is not drawn to scale. Arrows above (maternal allele) and below (paternal allele) exon 1 and the Zrsr1 gene represent the direction of transcription and the allelic expression status of the genes. The open and closed circles at the Zrsr1 promoter indicate unmethylation and methylation, respectively. A schematic of the targeting vector is shown under the gene. The closed and hatched boxes represent the truncation cassette and the neo-selection marker gene, respectively. These elements are flanked by the 5.2 kb left arm containing exon 1 and the 5.1 kb right arm, which contains part of intron 1. The truncation cassette is flanked by loxP sites, represented by gray arrowheads enclosed in open rectangles. Expected transcription patterns of the WT and PA alleles are shown above the gene schematic with thick lines and dotted lines corresponding to exons and introns, respectively. b RT-PCR analysis of Commd1 expression in growing oocytes prepared from B6 female neonates at Day 5 (D5), Day 10 (D10), and Day 15 (D15) postpartum, and fully grown MII oocytes (MII) from B6 adult females. PC: positive control for RT-PCR using adult brain cDNA. MW: molecular weight marker. c Analysis of methylation at Zrsr1 -DMR in growing and fully grown oocytes used in b . The 223-bp region in the DMR containing 14 CpGs was analyzed via bisulfite sequencing. Each row represents a dataset from one clone, and each circle represents one CpG site. Closed and open circles depict methylated and unmethylated CpGs, respectively

Article Snippet: Expression analysis To analyze Commd1 expression in oocyte and adult tissues, cDNA was synthesized with random primers (Takara, #3802) and reverse transcriptase (TOYOBO, TRT-101), and the Commd1 cDNA was amplified by PCR with the primers Comm-F1 (exon 1) and Comm-R1 (exon 2).

Techniques: Expressing, Methylation, Plasmid Preparation, Selection, Marker, Reverse Transcription Polymerase Chain Reaction, Positive Control, Molecular Weight, Methylation Sequencing

Journal: Epigenetics & Chromatin

Article Title: Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR

doi: 10.1186/s13072-018-0200-6

Figure Lengend Snippet: The antisense transcript in the promoter region of Commd1 in adult tissues. a Strand-specific RT-PCR was performed to detect an antisense transcript to Commd1 transcription in the promoter region. The arrows represent the primers, CommUP-F1, for cDNA synthesis, and CommUP-F2 and CommUP-R2, for subsequent RT-PCR. The amplicon is 206 bp in size. CommUP-R2 is located approximately 100-bp upstream from the putative transcription start site of Commd1 . b Brain (Br) and liver (Lv) RNA were analyzed. Tissues were prepared from adult F1 mice generated from crossing PA-B6 females with WT PWK males. cDNA synthesis was performed with (RTase +) or without (RTase −) reverse transcriptase. MW: molecular weight marker. c Allelic expression of the antisense transcripts was analyzed via direct sequencing of the amplified brain cDNA from the WT and PA mice shown in b . Three SNPs used to discriminate the parental alleles are shown under the electropherograms

Article Snippet: Expression analysis To analyze Commd1 expression in oocyte and adult tissues, cDNA was synthesized with random primers (Takara, #3802) and reverse transcriptase (TOYOBO, TRT-101), and the Commd1 cDNA was amplified by PCR with the primers Comm-F1 (exon 1) and Comm-R1 (exon 2).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Mouse Assay, Generated, Molecular Weight, Marker, Expressing, Sequencing

Journal: Cancer Cell International

Article Title: MicroRNA 200c-3p regulates autophagy via upregulation of endoplasmic reticulum stress in PC-3 cells

doi: 10.1186/s12935-017-0500-0

Figure Lengend Snippet: miR-200c-3p was associated with ER stress. a The level of miR-200c-3p was increased following thapsigargin (TG) treatment in PC-3 cells. After treatment with TG for 0, 1, 3, 6, 12, and 24 h, cells were lysed and complementary DNA was generated. RT-qPCR analysis was performed to determine the level of miR-200c-3p. U6 small nuclear ribonucleoprotein was used to normalize the expression of miR-200c-3p. b The expression levels of ATF6 and eukaryotic initiation factor (eIF)-2α were increased following TG treatment. RT-qPCR analysis was performed to evaluate the levels of ATF6 and elF2α. Levels of GAPDH were used for normalization. c Viability of PC-3 cells treated with TG in control or miR-200c mimics. Two days after transfection with miR-200c-3p mimic, 0.5 mM TG was added and cells were incubated for 48 h. The MTT assay was used to measure cell viability. Data are presented as the mean ± SEM of triplicate samples. ***p

Article Snippet: One microgram of total RNA was used to generate complementary DNA (cDNA) by superscript reverse transcriptase (Invitrogen).

Techniques: Generated, Quantitative RT-PCR, Expressing, Transfection, Incubation, MTT Assay

Journal: BMC Bioinformatics

Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

doi: 10.1186/1471-2105-6-107

Figure Lengend Snippet: Improved hierarchical clustering of combined NCI-60 cell-lines profiled by Affymetrix gene-chip and cDNA microarray by sequence-overlapping probe measurements. The gene expression profiles obtained for the sixty cell lines by the Affymetrix gene chips and the Stanford cDNA microarray platform were pooled after data transformation as described in the text. Gene expression data by the two different platforms were matched by either Unigene ID matching or by redefining the Affymetrix probe sets based on the sequence overlap criteria of the probes. The pooled gene expression profiles were subjected to average linkage hierarchical clustering. Matched cell-lines from the two platforms which cluster together are marked by red branches in the dendrogram. ( A ) Unigene-matched measurements tended to cluster the cell-lines by measurement platform, and produced only 28 instances of matched cell-lines clustering together. ( B ) Sequence-overlapping probe measurements produced more (43) instances of matched cell-lines from each platform clustering together.

Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

Techniques: Chromatin Immunoprecipitation, Microarray, Sequencing, Expressing, Transformation Assay, Produced

Journal: BMC Bioinformatics

Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

doi: 10.1186/1471-2105-6-107

Figure Lengend Snippet: Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HG-U95Av2 gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. ( A ), Affymetrix measurements matched to the cDNA centroids by Unigene identifier. ( B ), Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications. In particular, the large ERbB2+ subtype cluster (upper left) is mostly absent from the unigene-based classification. The significance of this cluster is supported by the observation that all tumors in this cluster for which Her-2 amplification was assessed by immunohistochemistry were designated positive.

Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

Techniques: Microarray, Chromatin Immunoprecipitation, Sequencing, Derivative Assay, Produced, Amplification, Immunohistochemistry

Journal: BMC Bioinformatics

Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

doi: 10.1186/1471-2105-6-107

Figure Lengend Snippet: Sequence-overlapping probes give greater cross-platform concordance for the NCI-60 panel. ( A ) Pearson correlation coefficient was calculated for each gene between its expression values measured on the Affymetrix Hu6800 platforms and its expression values measured on the Stanford cDNA microarray across sixty cell lines of the NCI-60 panel. The figure shows the cumulative distribution of the Pearson correlation coefficients for all genes analyzed. The five different curves reflect the level of cross-platform consistency of probe sets with various levels of overlap between the two microarray platforms. Matched gene measurements across the two platforms showed higher correlation when greater numbers of probes in the Affymetrix probe sets overlapped the insert region of the cDNA clone. The highest correlation was attained when only those Affymetrix probes overlapping the insert-sequence of a given cDNA clone were retained. Measurements for which the probes targeted the same transcript as the cDNA clone, but did not overlap the clone sequence, showed the lowest correlation. ( B ), Pearson correlation coefficient was calculated across all genes for each matched sample pair profiled by the Affymetrix Hu6800 platform and by the Stanford cDNA microarray. The figure shows the cumulative distribution of the Pearson correlation coefficients for the sixty cell lines of the NCI-60 panel. Matched cell-line measurements showed identical stratification of correlation levels by feature-matching criteria.

Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

Techniques: Sequencing, Expressing, Microarray

Journal: BMC Bioinformatics

Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

doi: 10.1186/1471-2105-6-107

Figure Lengend Snippet: Conserved clustering pattern of the NCI-60 cell lines profiled using cDNA microarray and Affymetrix gene chips. Data was normalized as described (methods). Average linkage Pearson correlation hierarchical clustering was computed for each dataset. Cell line names are colored according to cancer type.

Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

Techniques: Microarray

Journal: BMC Bioinformatics

Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

doi: 10.1186/1471-2105-6-107

Figure Lengend Snippet: Increased efficiency of breast cancer subtype classification transfer from cDNA microarray to Affymetrix HuFL gene-chip tumor-profiles by sequence-overlapping probe measurements. Tumor samples profiled on the Affymetrix platform were classified according to their correlation with the set of subtype median-centroids derived from cDNA microarray measurements (see methods). The classified samples were then hierarchically clustered using Pearson correlation and average-linkage agglomeration. Affymetrix measurements matched to cDNA centroids by sequence-overlap of probe features produced more coherent classifications than those obtained in the original transfer (Sørlie), specifically, more coherent Luminal A and ERBB2+ subtype clusters.

Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

Techniques: Microarray, Chromatin Immunoprecipitation, Sequencing, Derivative Assay, Produced

Journal: BMC Bioinformatics

Article Title: Redefinition of Affymetrix probe sets by sequence overlap with cDNA microarray probes reduces cross-platform inconsistencies in cancer-associated gene expression measurements

doi: 10.1186/1471-2105-6-107

Figure Lengend Snippet: Composition of redefined Affymetrix probe-sets based on overlap with cDNA clone insert sequence. Stacked histograms show the distribution of probe-set size for sets consisting of a single Affymetrix-defined probe-set (black) and for those comprised of probes originally grouped into separate probe-sets by Affymetrix (gray). A , NCI-60 10 k cDNA microarray to HuFL alternative CDF. B , Breast cancer 8 k cDNA microarray to HuFL alternative CDF. C , Breast cancer 8 k cDNA microarray to HG-U95Av2 alternative CDF. D , Lung cancer 22 k cDNA microarray to HG-U95Av2 alternative CDF.

Article Snippet: However, for a clear presentation of results we chose to compare the following classes representing different levels of shared identity: a) Affymetrix probe sets that share a Unigene ID with a cDNA clone. (termed Shared Unigene probes) b) Affymetrix probe sets containing probes that could be sequence-matched to the same transcript sequence as the cDNA clone, but for which no Affymetrix probe actually overlaps the cDNA clone sequence (termed Shared Transcript probes); c) Affymetrix probe sets with 1 to 10 probes sequence overlapping with the cDNA clone (termed Partially Overlapping probes); d) Affymetrix probe sets with 20 (i.e. all) probes sequence overlapping with the cDNA clone (termed Completely Overlapping probes); e) alt-CDF or "redefined probe sets" for which all probes across the entire array that matched to a given cDNA clone insert were used to define a new derivative probe set.

Techniques: Sequencing, Microarray

Journal: PLoS Neglected Tropical Diseases

Article Title: Identification of a host collagen inducing factor from the excretory secretory proteins of Trichinella spiralis

doi: 10.1371/journal.pntd.0006516

Figure Lengend Snippet: Expression levels of type I collagen, Smad2/3, and TGF-β1 genes after ES-P treatment in aged mice. (A) The left ears of fourteen-week old mice were treated daily with 30 μg T . spiralis ES-P for 14 days (red color). The right ears of were treated daily with PBS for 14 days as control (blue color). After 14 days, ears were collected. Total RNA was extracted from each ear tissue and cDNA was constructed. (B) The gene expression levels of collagen I , TGF-β1 , and Smad2/3 were analyzed by real-time PCR. The gene expression value of each group was normalized by control value, 14 weeks group. (**; P

Article Snippet: Real-time PCR Homogenized ear tissues were mixed with TRIzol (Invitrogen, Germany), and total RNA extraction and cDNA synthesis (Invitrogen, Germany) was performed in accordance with the manufacturer’s protocols.

Techniques: Expressing, Mouse Assay, Construct, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS

doi: 10.1038/srep19118

Figure Lengend Snippet: The role of TDP-43 in the expression of VHL, CUL2, and HIF1α. ( a–f ) Measurement of VHL in the presence of overexpression ( a,c,d ) or knock down ( b,e,f ) of TDP-43 in HEK293A cells. ( a,b ) Quantitative real-time PCR analysis of VHL expression in the presence of overexpression or knock down of TDP-43. HEK293A cells were transiently transfected with WT or mutant NLS TDP-43 ( a ) or siRNA oligonucleotides targeting TDP-43 ( b ). At 48 h and 96 h after transfection in ( a ) and ( b ), respectively, cells were harvested for cDNA generation from RNA. In ( b ), plasmid for WT TDP-43 was additionally introduced at 48 h before harvest for the rescue experiment. Each data point represents the average from three ( a ) and six ( b ) cultures (mean ± SD; *p

Article Snippet: Quantitative real-time PCR The total RNA samples were purified from the transfected cells using a commercially available kit (Invitrogen) and were converted to cDNA with reverse transcriptase (Toyobo, Tokyo, Japan).

Techniques: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Transfection, Mutagenesis, Plasmid Preparation

Journal: Viruses

Article Title: Neurotropism In Vitro and Mouse Models of Severe and Mild Infection with Clinical Strains of Enterovirus 71

doi: 10.3390/v9110351

Figure Lengend Snippet: Enterovirus 71 (EV71) strain infection and replication in rhabdomyosarcoma (RD) cells, mouse neuroblastoma Neuro-2a (N2a) cells and human neuroblastoma (SK-N-SH) cells. ( A ) Images of RD, N2a, and SK-N-SH cells acquired 24 hours post infection (hpi) after infection with 100 50% tissue culture infective dose (TCID50) of three representative EV71 strains, including clinical mild strain Hun11-4, clinical severe strain NX10-36, and clinical fatal strain GD10-45 (magnification 200×). ( B ) Replicative curve of EV71 in RD cells. Total RNA was prepared from infected and uninfected RD cells at 2, 6, 12, 24, 48, and 72 hpi. After reverse transcription, VP-1 specific primers were used to amplify the viral VP1 gene transcript from the complementary DNA (cDNA), confirming viral replication in infected RD cells. ( C ) Replicative curve of EV71 in SK-N-SH cells. Total RNA was prepared from infected and uninfected SK-N-SH cells at 2, 6, 12, 24, 48, and 72 hpi. After reverse transcription, VP-1 specific primers were used to amplify the viral VP1 gene transcript from the cDNA, confirming viral replication in infected SK-N-SH cells.

Article Snippet: The polymerase chain reaction (PCR) parameters for all primer pairs were as follows: complementary DNA (cDNA) was denatured at 94 °C for 5 min. Amplification was performed using KOD-plus-DNA Polymerase (Toyobo, Tokyo, Japan) with 35 cycles of denaturation for 30 s at 94 °C, primer annealing for 45 s at 56 °C, and elongation for 1 min at 68 °C, followed by extension at 68 °C for 10 min.

Techniques: Infection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease

doi: 10.1073/pnas.1620299114

Figure Lengend Snippet: Increased expression of CRRY in the RPE cells by subretinal injection of AAV-CRRY. ( A ) Histogram showing relative CRRY, DAF1, DAF2, CD59a, CD59b, and CFH mRNA levels by qRT-PCR. Each mRNA level was normalized to 18S rRNA. ( B ) Representative immunoblot of CRRY (65-kDa isoform), Myc, and β-actin using RPE homogenate (10 μg of protein per lane). Protein and cDNA samples were obtained from 1-y-old Abca4 −/− mice injected with AAV-CRRY and -null viruses. Data represent mean ± SD; n = 5 mice. Each protein and cDNA sample was run in triplicate. M.W., molecular weight.

Article Snippet: The murine CRRY gene was amplified by PCR from a cDNA clone (Origene ) ( ).

Techniques: Expressing, Injection, Quantitative RT-PCR, Mouse Assay, Molecular Weight

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease

doi: 10.1073/pnas.1620299114

Figure Lengend Snippet: AAV-CRRY treatment does not change the oxidative stress level in Abca4 −/− mice. ( A ) Histogram showing the relative SOD-1 and SOD-2 mRNA levels by qRT-PCR. Each mRNA level was normalized to 18S rRNA. ( B ) Representative immunoblots of SOD-1, SOD-2, and α-tubulin using RPE homogenate (10 μg of protein per lane). ( C ) Histogram showing SOD-1 and SOD-2 normalized protein data in the RPE homogenate samples shown in B . Protein and cDNA samples were obtained from 7-mo-old Abca4 −/− mice injected with AAV-CRRY and -null viruses ( n = 4). Each protein and cDNA sample was run in triplicate.

Article Snippet: The murine CRRY gene was amplified by PCR from a cDNA clone (Origene ) ( ).

Techniques: Mouse Assay, Quantitative RT-PCR, Western Blot, Injection

Journal: Nucleic Acid Therapeutics

Article Title: Multiple microRNAs Derived from Chemically Synthesized Precursors Regulate Thrombospondin 1 Expression

doi: 10.1089/nat.2013.0467

Figure Lengend Snippet: Inverse correlation between THBS1 and TGF-β1 through miRNAs. TGF-β1 overexpression by cDNA reduced THBS1 and upregulated let-7a, let-7b, and miR-18a. (A) HeLa cells were transfected with TGF-β1 complementary DNA (0 and 60 ng)

Article Snippet: HeLa cells were transfected with 60 ng complementary DNA (cDNA) of TGF-β1 (SC119746) from OriGene using jetPEI in 24-well plates.

Techniques: Over Expression, Transfection

Journal: Genome Biology

Article Title: scDual-Seq: mapping the gene regulatory program of Salmonella infection by host and pathogen single-cell RNA-sequencing

doi: 10.1186/s13059-017-1340-x

Figure Lengend Snippet: A single-cell RNA-sequencing approach to studying host–pathogen interaction. a Heterogeneity of outcomes of intracellular infection is due to both Salmonella and macrophage states. scDual-Seq simultaneously produces the transcriptome of both the host and the pathogen and allows the identification of cellular subpopulations during infection. b Schematic of the scDual-Seq method. Reverse transcription is primed using random hexamers, followed by RNase treatment and 3’ polyA tailing. The second strand is synthesized using the CEL-Seq2 barcoded primers (see “ Methods ”). The samples are pooled together before the complementary DNA (cDNA) undergoes linear amplification by in vitro transcription. The amplified RNA is then reverse transcribed using a random primer with an overhang of the sequence complementary to the Illumina 3’ adaptor. cDNA with both Illumina adaptors are selected by polymerase chain reaction and the DNA library is sequenced using paired-end Illumina sequencing. c Mean number of unique transcripts identified across five technical replicates, for mouse ( black ) and Salmonella ( red ). Circles and error bars represent the mean and standard deviation. d Plot between the expression of the two technical replicates of 10 pg mouse RNA and 10 pg Salmonella RNA. e Boxplots indicating the correlation coefficients across replicates with the sum expression of all 20 samples for mouse and for five replicates in each dilution for Salmonella . Mouse indicated in black , Salmonella dilutions indicated in red

Article Snippet: After adding a polyA tail to the complementary DNA (cDNA), scDual-Seq proceeds as an extensive modification of the CEL-Seq2 method for single-cell RNA-seq [ , ], which includes barcoding for multiplexing, IVT for amplification, and paired-end Illumina sequencing.

Techniques: RNA Sequencing Assay, Infection, Synthesized, Amplification, In Vitro, Sequencing, Polymerase Chain Reaction, Standard Deviation, Expressing