Journal: World Journal of Gastroenterology : WJG

Article Title: p73 G4C14 to A4T14 polymorphism is associated with colorectal cancer risk and survival

doi: 10.3748/wjg.v16.i35.4448

Figure Lengend Snippet: Detection of the p73 genotype by polymerase chain reaction with confronting two-pair primers. Lane M: 100-bp DNA ladder; Lanes 1 and 5: GC/GC genotype; Lanes 2, 3 and 6: GC/AT genotype; Lane 4: AT/AT genotype.

Article Snippet: PCR was performed in a volume of 20 μL containing 100 ng of DNA template, 0.2 mmol/L each deoxynucleotide triphosphate, 1 × PCR buffer (50 mmol/L KCl, 10 mmol/L Tris HCl, and 0.1% Triton X-100), 1.5 mmol/L MgCl2 , 1 U of Taq polymerase (Sigma-Aldrich Biotechnology) and 10 pmol of each of four primers.

Techniques: Polymerase Chain Reaction

Journal: Journal of Ayurveda and Integrative Medicine

Article Title: DNA barcoding of authentic and substitute samples of herb of the family Asparagaceae and Asclepiadaceae based on the ITS2 region

doi: 10.4103/0975-9476.100177

Figure Lengend Snippet: PCR for the ITS2 region of root DNA samples of family Asparagaceae ( Asparagus recemosus and Asparagus gonoclados ) and Asclepiadaceae ( Hemidesmus indicus and Decalepis hamiltonii ) Detection of the ITS2 region of ~500 bp PCR product on a 1.5% agarose gel. Lane 1. Asparagus racemosus , Lane 2. Asparagus gonoclados , Lane 3. Hemidesmus indicus , Lane 4. Decalepis hamiltonii , Lane 5.100 bp ladder, Lane 6. Negative control

Article Snippet: [ ] PCR amplification was performed in 25 μl reaction mixtures containing approximately 40 ng template DNA, 1 X PCR buffer, 1.5 mM MgCl2 , 0.2 mM of each dNTP, 0.1 μM of each primers (Sigma Proligo and Sigma Life Science, India), and 1.0 U Taq DNA Polymerase (Invitrogen) in a thermal cycler (Eppendorf, Germany).

Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control

Journal: Journal of Transplantation

Article Title: Inflammation-Mediated Regulation of MicroRNA Expression in Transplanted Pancreatic Islets

doi: 10.1155/2012/723614

Figure Lengend Snippet: Overexpression of miR-21 regulates endogenous Pdcd4 and Pclo mRNAs. (a) MIN6 cells were treated 24 hours with cytokine cocktail IL-1 β (50 U/mL), TNF- α (2000 U/mL), and IFN γ (100 U/mL). The expression of miR-21 was assessed by qRT-PCR. (b) Overexpression of miR-21 mimic (300 nM) for 48 hs inhibits the expression of endogenous Pdcd4 and Pclo mRNA. Experiments shown in (a) and (b) are expressed as mean ± SD ( n = 5), * P

Article Snippet: The isolated RNA can be used for miRNA as well as mRNA analysis. cDNA synthesis and PCR amplification were performed according to the manufacturer's protocol (Applied Biosystems).

Techniques: Over Expression, Expressing, Quantitative RT-PCR

Journal: Journal of phycology

Article Title: Phylogenetic analysis of LSU and SSU rDNA group I introns of lichen photobionts associated with the genera Xanthoria and Xanthomendoza (Teloschistaceae, lichenized Ascomycetes)

doi: 10.1111/jpy.12126

Figure Lengend Snippet: DNA extraction, PCR amplification and sequencing

Article Snippet: The PCR reactions were performed in 50 μL reaction volumes containing a reaction mix of 0.2 mM of each of four dNTPs, 1.5 μM of each PCR primer, and 1.25 U of Taq DNA polymerase (Sigma Aldrich, Buchs, SG, Switzerland) and 5 μL of 10X PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2 and 0.01% gelatin).

Techniques: DNA Extraction, Polymerase Chain Reaction, Amplification, Sequencing

Journal: Journal of Pest Science

Article Title: Diagnostic PCR assays to unravel food web interactions in cereal crops with focus on biological control of aphids

doi: 10.1007/s10340-015-0685-8

Figure Lengend Snippet: Gel image of PCR products amplified with the three customised multiplex PCR assays and visualised with the QIAxcel system. MPI ( left side ) comprises group/family-specific primers for seven taxa: beetles/thrips, spiders, aphids, earthworms, springtails, dipterans, and lacewings. MPII spiders ( upper right side ) covers two spider families, i.e. lycosids and linyphiids, as well as the genus Pachygnatha . MPII beetles/thrips ( lower right side ) addresses six taxa: the carabid genera Poecilus , Bembidion , Pterostichus, and Harpalus , the ladybeetle Coccinella septempunctata as well as thrips ( Frankliniella , Limothrips ). The shortest and longest fragments within each lane represent the two alignment markers (AM; 15 and 3000 bp) as indicated in the left panel . For amplicon lengths see Fig. 1 and Table 3 . Mixes of DNA templates of targets; approximately 1000 ds copies each in PCR

Article Snippet: The PCR protocol of the MPII beetles/thrips assay differed only slightly from the MPII spiders : 1.5 µl of DNA extract and 30 mM TMAC (Sigma-Aldrich) were used in the total volume of 10 µl (plus PCR-grade water to adjust the volume); thermocycling conditions as described above, but with an annealing temperature of 63.5 °C.

Techniques: Polymerase Chain Reaction, Amplification, Multiplex Assay

Journal: PLoS ONE

Article Title: Inducible Expression of the De-Novo Designed Antimicrobial Peptide SP1-1 in Tomato Confers Resistance to Xanthomonas campestris pv. vesicatoria

doi: 10.1371/journal.pone.0164097

Figure Lengend Snippet: Transgenic tomato Micro Tom lines expressing the AMP SP1-1. (A) Schematic illustration of PMIGW-4XW2/4XS::SP1-1 vector construct. The vector contains a CaMV35S promoter-driven phosphomannose isomerase (PMI) gene for mannose selection. B1, B2, B3 and B4 represent attB Gateway recombination sites. SP, signal peptide RsAFP1 from radish; SP1-1, synthetic antimicrobial peptide; TNos, nopaline synthase gene terminator; RB, right border; LB, left border; W2 cis -acting elements from the parsley PR1 gene; S, cis -elements from the parsley EL17 gene; CaMV35S, minimal promoter containing the sequence -46 to +8 from the cauliflower mosaic virus 35S promoter; T35S, terminator from the cauliflower mosaic virus 35S. (B) Molecular characterization of transformed plants. PCR was done using DNA from young leaves as template and RsAFP1-TNos-specific primers. PCR fragments were obtained for T583-4, T583-5 and T583-6. PMIGW-4XW2/4XS::SP1-1 transformation vectors and WT Micro Tom tomato plants were used as positive and negative controls respectively. (C and D) Morphological phenotypes of six weeks old WT and transgenic T1 Micro Tom tomato plants. Transgenic and WT plants were grown in climate chambers for 6 weeks. (E) Detached leaves of line T583 were infiltrated with water (H) or pep25 peptide (P). Total RNA was extracted before induction (BI) and 0, 3, and 22 h after induction. RT-PCR was carried out with gene-specific primers for RsAFP1-SP1-1 and actin and fragments of 163 bp and 586 bp were expected, respectively. Genomic DNA of the transgenic line T583 was used as control (+). Total RNA from WT plants and water (-) was used as negative control. The SP1-1 band is marked with an arrow. M, 100 bp DNA ladder.

Article Snippet: PCR amplifications from genomic DNA were done using the PCR super mix (Invitrogen) following the manufacturer´s instructions.

Techniques: Transgenic Assay, Expressing, Plasmid Preparation, Construct, Selection, Sequencing, Transformation Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control

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

Article Title: Transcriptome profiling of Plasmodium vivax in Saimiri monkeys identifies potential ligands for invasion

doi: 10.1073/pnas.1818485116

Figure Lengend Snippet: Validation of RNAseq data by qRT-PCR. ( A ) qRT-PCR was performed on eight genes from P. vivax Sal I infection in Saimiri and Aotus infection. The normalized mRNA level (averaged) and SD for all eight genes [PVX_099980 (MSP1), PVX_110810 (DBP1), PVX_094230 (hypothetical protein), PVX_01695 (Pf-fam-b), PVX_104690 (Pv-fam-c), PVX_097670 (MSP3g), PVX_092275 (AMA1), and PVX_112690 (Pv-fam-a)] were graphed for each Saimiri and Aotus monkey sample. Each gene signal was normalized to the housekeeping gene GST. Each bar represents an average of two monkeys. The error bar is based on the SD. ( B ) Table shows the correlation of qRT-PCR and the RNAseq data with the number of samples ( n = 4) and P value (significance of correlation).

Article Snippet: Briefly, Life Technologies Express qRT-PCR Supermix with premixed ROX dye (ThermoFisher Scientific) was used to perform the assays.

Techniques: Quantitative RT-PCR, Infection

Journal:

Article Title: A patterned anisotropic nanofluidic sieving structure for continuous-flow separation of DNA and proteins

doi: 10.1038/nnano.2006.206

Figure Lengend Snippet: O Ogston sieving of short DNA (the PCR marker) through the ANA

Article Snippet: The PCR marker as well as λ DNA–Hind III digest (New England BioLabs, Beverly, MA) were labeled with the intercalating fluorescence dye YOYO-1 (Molecular Probes, Eugene, OR) in TBE 5× buffer.

Techniques: Polymerase Chain Reaction, Marker

Journal: Scientific Reports

Article Title: Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition

doi: 10.1038/s41598-018-19930-w

Figure Lengend Snippet: Radiation induced mitochondrial biogenesis augments metabolic viability: ( A ) Cell cycle histogram showing phase distribution (G1, S and G2/M) of cells at 24 and 48 hrs post irradiation in HeLa and MDA-MB-231 cells. ( B ) Mitochondrial genome encoded Leu tRNA gene was analyzed by semi quantitative PCR and normalized with nuclear pol gamma gene copy number. The mtDNA copy number is also presented as comparative fold change at respective time points (bar diagram). ( C ) Protein expression analysis of mitochondrial biogenesis and mitochondrial complex-II subunit SDH-A presented in HeLa and MDA-MB-231 cells. The values between the blots represent the fold increase at 8 and 24 hrs. post irradiation quantified by densitometry and normalized with respective β-Actin. The DNA ( B ) and protein blot ( C ) images were cropped from full-length blots (Supplementary Figs 2 and 3 ). ( D ) Analysis of effect of chloramphenicol (40 μM; 30 mins prior to IR; continuous exposure) on mitochondrial content by MitoTracker Green FM at indicated time points using flow cytometer and graphs presented as fold change of mean fluorescence intensity (MFI) with respective control. Effect of Chloramphenicol on radiation induced growth inhibition was analyzed (at 5 Gy) by MTT assay ( E ) and cell number ( F ) in HeLa and MDA-MB-231 cells. Growth inhibition quantified and mentioned at 48 hours. Data are expressed as mean ± SD from triplicates. *p

Article Snippet: Primers were purchased from GCC biotech (India) while PCR master mix and hot start DNA polymerase was procured from Sigma, USA.

Techniques: Irradiation, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Fluorescence, Inhibition, MTT Assay

Journal: The Journal of Veterinary Medical Science

Article Title: The widely distributed hard tick, Haemaphysalis longicornis, can retain canine parvovirus, but not be infected in laboratory condition

doi: 10.1292/jvms.14-0199

Figure Lengend Snippet: Detection of FeLV gene from the FeLV-inoculated unfed adult ticks by RT-PCR. The tick actin mRNA was detected and amplified as a loading control. The numbers indicate each dpi, and each dpi consists of 3 individual tick samples. N, negative control ticks; P, undiluted FeLV stock solution.

Article Snippet: RNA extraction and reverse-transcriptase PCR (RT-PCR) for FeLV : Homogenized samples were added with TRI® reagent (Sigma-Aldrich).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control

Journal: Microbial Cell Factories

Article Title: Lactic acid fermentation as a tool to enhance the functional features of Echinacea spp

doi: 10.1186/1475-2859-12-44

Figure Lengend Snippet: Expression of TNF-α gene of Caco-2 cells. The expression was analyzed after treatment with Echinacea suspension (ES) (1-50 μg/ml) for 16 ( A ), 24 ( B ), and 48 h ( C ), as determined by RT-PCR. ES fermented for 24 h at 30°C with Lactobacillus plantarum 1MR20 (1MR20-ES), C2 (C2-ES) and POM1 (POM1-ES), and with the association between strains 1MR20 and C2 (1MR20/C2-ES) were assayed. ES-CT, chemically acidified ES, without bacterial inoculum, and incubated for 24 h at 30°C, was the control. Results were expressed as percent ratio to lipopolysaccharide (5 μg/ml) (LPS)-treated cells (LPS). Data are the means of three independent experiments twice analysed.

Article Snippet: Freeze dried ES at the concentrations of 1, 10, and 50 μg/ml was added to 80% confluent Caco-2 cells with LPS (5 μg/ml), and incubated at 37°C for 16, 24 and 48 h. For quantitative real-time PCR (RT-PCR), total RNA from Caco-2 cells was extracted using Tri Reagent (Sigma Aldrich), as described by Chomczynski and Mackey [ ].

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation

Journal: Scientific Reports

Article Title: Application of IL-36 receptor antagonist weakens CCL20 expression and impairs recovery in the late phase of murine acetaminophen-induced liver injury

doi: 10.1038/srep08521

Figure Lengend Snippet: CCL20 expression in murine APAP-induced liver injury. (a) Mice received PBS (n = 6) or APAP (6 h, n = 6; 24 h, n = 9; 48 h, n = 11) for indicated time points. Hepatic CCL20 and IL-36γ mRNA was determined by realtime PCR. Target mRNA was normalized to that of GAPDH (means ± SEM versus ctrl of target mRNA, *** p

Article Snippet: Analysis of cytokine expression by realtime PCR After RNA isolation (Tri-Reagent, Sigma-Aldrich), 1 μg of total RNA was transcribed using hexameric primers and Moloney virus RT (Life Technologies).

Techniques: Expressing, Mouse Assay, Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Application of IL-36 receptor antagonist weakens CCL20 expression and impairs recovery in the late phase of murine acetaminophen-induced liver injury

doi: 10.1038/srep08521

Figure Lengend Snippet: Expression of IL-36γ in murine APAP-induced liver injury and inflamed hepatocytes. (ab) Mice received PBS (n = 6) or APAP or (where indicated) APAP/IL-22 (6 h (n = 6), 24 h (n = 9)). (a) Hepatic IL-36γ mRNA was determined by realtime PCR. Target mRNA was normalized to that of GAPDH (means ± SEM versus ctrl; ** p

Article Snippet: Analysis of cytokine expression by realtime PCR After RNA isolation (Tri-Reagent, Sigma-Aldrich), 1 μg of total RNA was transcribed using hexameric primers and Moloney virus RT (Life Technologies).

Techniques: Expressing, Mouse Assay, Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Application of IL-36 receptor antagonist weakens CCL20 expression and impairs recovery in the late phase of murine acetaminophen-induced liver injury

doi: 10.1038/srep08521

Figure Lengend Snippet: Application of IL-36Ra modulates CCL20 expression in late murine APAP-induced liver injury. (abc) Mice received APAP (n = 11) or APAP/IL-36Ra (n = 12) and were maintained for 48 h. Thereafter, hepatic CCL20 mRNA (a) and protein (b) was determined by realtime PCR and ELISA, respectively. (a) Target mRNA was normalized to that of GAPDH and is shown as percent of APAP alone (means ± SEM). (b) Liver tissue CCL20 content was determined by ELISA, is depicted as pg/500 μg total protein, and shown as means ± SEM. (c) Hepatic CCL2 and CXCL2 mRNA was determined by realtime PCR. Target mRNA was normalized to that of GAPDH and is shown as percent of APAP alone set as 100% (means ± SEM). Statistical analysis, Student's t-test; n.s., not significant.

Article Snippet: Analysis of cytokine expression by realtime PCR After RNA isolation (Tri-Reagent, Sigma-Aldrich), 1 μg of total RNA was transcribed using hexameric primers and Moloney virus RT (Life Technologies).

Techniques: Expressing, Mouse Assay, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay