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: 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: Blood Cancer Journal

Article Title: Novel function of FAXDC2 in megakaryopoiesis

doi: 10.1038/bcj.2016.87

Figure Lengend Snippet: FAXDC2 expedites megakaryopoiesis in murine bone marrow cells. ( a ) c-kit-positive progenitor cells isolated from primary murine bone marrow cells were transduced with control lentiviral vector (Ctrl) or FAXDC2-overexpressing (FAXDC2) vector to undergo megakaryocytic differentiation with thrombopoietin (TPO) for 6 days. The resultant cells were stained with phycoerythrin (PE)-CY7-conjugated anti-CD41 antibody and analyzed by flow cytometry (left panel). Bar graph (right panel) was the statistics of left panel. ( b ) The resultant cells were stained with PE-conjugated anti CD42 antibody and analyzed by flow cytometry (left panel). Bar graph (right panel) was the statistics of left panel. ( c ) The resultant cells were stained with anti-CD41-PE-CY7 and DAPI. The CD41 + cells were gated for analysis of DNA content. The DNA content that is > 4N is represented (left panel). Bar graph (right panel) was the statistics of left panel. ( d ) The control (Ctrl) or FAXDC2-overexpressing (FAXDC2) cells were harvested for Wright-Giemsa stains. The stained cells were photographed under microscopy at the bright view of the microscope (magnification × 20 or magnification × 40). Scale Bar: 100 μm. ( e ) The control (Ctrl) or FAXDC2-overexpressing (FAXDC2) cells were harvested for quantitative real-time PCR (RT-PCR) at mRNA level. The expression of RUNX1 was measured. * P

Article Snippet: RNA preparation and quantitative real-time PCR Total RNA was extracted with Trizol and complementary DNA was prepared with reverse transcriptase by standard procedure following instructions of the manufacturer (Invitrogen, Grand Island, NY, USA).

Techniques: Isolation, Transduction, Plasmid Preparation, Staining, Flow Cytometry, Cytometry, Microscopy, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Genes

Article Title: Deep Transcriptome Sequencing of Two Green Algae, Chara vulgaris and Chlamydomonas reinhardtii, Provides No Evidence of Organellar RNA Editing

doi: 10.3390/genes8020080

Figure Lengend Snippet: Alignment of the deep sequenced mitochondrial transcriptome of Chlamydomonas reinhardtii strain cc503. ( A ) A single putative RNA editing site was detected in the cytochrome oxidase b gene at nucleotide 1,182. The mean coverage of the cob gene was 28,371.5 ± 7,598 with a min:max of 816:42,177. ( B ) Sequencing of the cob DNA and cDNA from the cc503 isolate used for the deep sequencing revealed a SNP at that site, therefore no editing had taken place.

Article Snippet: Complementary DNA (cDNA) was produced from DNase treated RNA using Bio-Rad’s iScript cDNA synthesis kit (Hercules, CA, USA).

Techniques: Sequencing

Journal: Genes

Article Title: Deep Transcriptome Sequencing of Two Green Algae, Chara vulgaris and Chlamydomonas reinhardtii, Provides No Evidence of Organellar RNA Editing

doi: 10.3390/genes8020080

Figure Lengend Snippet: Three follow-up experiments attempting to detect three putative RNA editing sites in the C. vulgaris psbI mRNA. ( A ) Chara was grown in nine different environmental conditions: 20 °C, 25 °C, 30 °C and pH 6, 7, 8, in triplicate, and then DNA + RNA were extracted from each. The melt temperatures for genomic DNA (gDNA) and complementary DNA (cDNA) were determined as paired reactions using high resolution melt analysis. Melt temperature differences between gDNA and cDNA derived amplicons were calculated by subtraction. Data presented on these graphs are the averages of nine observations ± standard error (SE). There were qualitative differences in the cDNA–DNA melt temperatures of psbI and the negative control, psbA , at the different incubation temperatures but these differences were statistically insignificant. Different pH also had no significant effect on the cDNA–DNA melt temperatures and two-way analysis of variance detected no interaction between the temperature and pH for either gene; ( B ) RNase H-dependent PCR (rhPCR) was used in an attempt to detect single base changes between the psbI gDNA and cDNA extracted from Chara incubated in different temperatures. This assay was designed to suppress PCR amplification of the genomic sequence but not cDNA made from an edited transcript. The quantitative PCR (qPCR) amplification curves show that gDNA and cDNA amplified with sigmoidal curves with standard PCR but that both gDNA and cDNA were suppressed by the addition of an rhPCR primer despite the presence of RNase H2 in the reactions. This suggests no editing occurred at any of the tested temperatures; ( C ) cDNAs were PCR amplified and sequenced from total RNA extracted from Chara grown at 20 °C, 25 °C, and 30 °C. The sequences all matched the genomic sequence, suggesting no editing had taken place.

Article Snippet: Complementary DNA (cDNA) was produced from DNase treated RNA using Bio-Rad’s iScript cDNA synthesis kit (Hercules, CA, USA).

Techniques: Derivative Assay, Negative Control, Incubation, RNase H-dependent PCR, Polymerase Chain Reaction, Amplification, Sequencing, Real-time Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: A σE-Mediated Temperature Gauge Controls a Switch from LuxR-Mediated Virulence Gene Expression to Thermal Stress Adaptation in Vibrio alginolyticus

doi: 10.1371/journal.ppat.1005645

Figure Lengend Snippet: RpoE binds to target DNA in response to temperature changes in vivo and in vitro . ( A ) ChIP assays were used to analyze RpoE binding to the luxR , rpoE and rpoH promoter in vivo . Cells were cultured at different temperatures for 9 h. They were then cross-linked, washed, and sonicated to produce sheared chromosomal DNA was purified from the sheared pellets both before precipitation (input) and after precipitation in the presence (+) and absence (-) of the anti-RpoE antibody (IP). The DNA was then amplified using PCR with the primers P luxR chip-F/R, P rpoE -chipF/R, P rpoH -chipF/R and control-F/R ( S2 Table ). ( B ) ChIP assays were followed by qPCR to determine the relative enrichment in DNA molecules that were bound to RpoE at different temperatures. The results are shown normalized to the control gene gyrB . Results were calculated using the ΔΔ C T method. * P

Article Snippet: Quantitative real-time reverse transcription PCR (qRT-PCR) Equal amounts of RNA (1 μg) were used to generate complementary DNA (Toyobo, Tsuruga, Japan) using random primers.

Techniques: In Vivo, In Vitro, Chromatin Immunoprecipitation, Binding Assay, Cell Culture, Sonication, Purification, Amplification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: A σE-Mediated Temperature Gauge Controls a Switch from LuxR-Mediated Virulence Gene Expression to Thermal Stress Adaptation in Vibrio alginolyticus

doi: 10.1371/journal.ppat.1005645

Figure Lengend Snippet: RpoE binds directly to the luxR promoter region. ( A ) EMSAs were performed with purified RpoE, and the luxR promoter region was analyzed. The amount of RpoE protein (nM) that was used is indicated, and 20 ng of each Cy5-labelled probe was added to the EMSA reactions. The shifts were verified to be specific in experiments in which we added a 5- to 50-fold excess of unlabeled specific DNA and non-specific competitor DNA (poly(dI:dC)). ( B ) Plot showing the affinity of RpoE to the luxR promoter. The densitometric intensities of bound DNA fragments were plotted against RpoE concentrations. The arrow indicates the concentration of RpoE that caused half-maximal binding ( K d ). ( C )Footprinting analysis of RpoE binding to a binding site in the luxR promoter. Electropherograms of a DNase I digest of the P luxR promoter probe (400 ng) after incubation with 0 or 400 nM RpoE. The respective nucleotide sequences that were protected by His-RpoE are indicated below, and the specific -10 and -35 regions are underlined. ( D-E ) In vitro transcription was performed using a P rpoE template, ( D ) a P luxR template ( E ), NTP, and RNAP core enzyme as well as RpoE. Various concentrations of purified LuxR were added into the reaction mixture to determine its effect on luxR transcription ( E ). The transcripts were purified, reverse-transcribed (RT, +) and detected using PCR. As a control, the same purified transcripts were also treated using the same process but without reverse transcriptase (RT, -).

Article Snippet: Quantitative real-time reverse transcription PCR (qRT-PCR) Equal amounts of RNA (1 μg) were used to generate complementary DNA (Toyobo, Tsuruga, Japan) using random primers.

Techniques: Purification, Concentration Assay, Binding Assay, Footprinting, Incubation, In Vitro, Polymerase Chain Reaction

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: 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

Journal: Reproductive Sciences

Article Title: Regulation of sPLA2-IID in Human Decidua

doi: 10.1177/1933719113519176

Figure Lengend Snippet: Representative reverse transcriptase–polymerase chain reaction (RT-PCR) gel showing expression of sPLA 2 -IID (394 bp) and the reference gene glyceraldehyde phosphate dehydrogenase ( GAPDH ; 598 bp) in pooled complementary DNA (cDNA; pooled from n

Article Snippet: RNA was subsequently isolated using Qiagen RNeasy kits (Qiagen Inc) and 100 ng was used to synthesize complementary DNA (cDNA) with qScript cDNA SuperMix (Quanta BioSciences, Gaithersburg, Maryland).

Techniques: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Insects

Article Title: Impact of Subolesin and Cystatin Knockdown by RNA Interference in Adult Female Haemaphysalis longicornis (Acari: Ixodidae) on Blood Engorgement and Reproduction

doi: 10.3390/insects9020039

Figure Lengend Snippet: Detection of salivary cystatin (HlSC-1) and subolesin by semi-quantitative RT-PCR. Salivary glands were collected from adult female H. longicornis which had been fed for five days. Total RNA was extracted, and a cDNA synthesis was performed. An RT-PCR was performed using gene-specific primers. Lane 1 indicates a 100 bp DNA ladder; lane 2, subolesin 396 bp; lane 3, cystatin 300 bp; and lane 4, actin 540 bp.

Article Snippet: Complementary DNA (cDNA) was synthesized using a transcriptor first-strand cDNA synthesis kit (Roche Holding AG, Basel, Switzerland) in accordance with the manufacturer’s instructions, using 1 µg of total RNA and an anchored oligo (dT)18 primer.

Techniques: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Therapy. Nucleic Acids

Article Title: Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1

doi: 10.1038/mtna.2016.93

Figure Lengend Snippet: Pyrosequencing assay for detection of correctly spliced transcript in fibroblasts of patients with the c.610 + 364G > A mutation . The N158S-forward primer binds in exon 3, the N158S-biot-reverse primer at the junction of exon 4b and exon 5. This primer setting exclusively amplifies cDNA of correctly spliced OPA1 transcripts (top bar, wildtype allele) but no transcripts with retained cryptic exon c between exons 4b/5 (middle bar, mutant allele, and upper pyrosequencing scheme). The sequencing primer (N158S-seq) binds two bases upstream of the heterozygous SNP rs7624750 in exon 4. The A allele of rs7624750 cosegregates with the mutation and cannot be detected in cDNA from mutant untreated cells using this assay. Upon AON treatment, cryptic exon c is skipped and the mutant transcript can be amplified (lower bar) and hence its allelic contribution can be measured via pyrosequencing (lower left scheme). A relative ratio of 35.3% versus 64.7% (A-allele versus G-allele) corresponds to a rescue of 55% of all mutant alleles in the cDNA of treated fibroblasts (example), which corresponds to a shift of total correctly spliced OPA1 mRNA of 77.5% (lower graph) For further graphical illustration of the principle compare Supplementary Figure S2 . AON, antisense oligonucleotides; cDNA, complementary DNA.

Article Snippet: Cells were harvested at different time points, using the Peq Gold total RNA kit (Peqlab, Erlangen, Germany), followed by complementary DNA (cDNA) synthesis with the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Mannheim, Germany).

Techniques: Pyrosequencing Assay, Mutagenesis, Sequencing, Amplification