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: 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: 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