Journal: Molecular Plant Pathology

Article Title: Secondary amplification of siRNA machinery limits the application of spray‐induced gene silencing

doi: 10.1111/mpp.12728

Figure Lengend Snippet: The duration of the effect of RNA interference (RNAi) in Fusarium asiaticum cells. Fusarium asiaticum was grown in SNA medium (0.1% KH 2 PO 4 , 0.1% KNO 3 , 0.05% MgSO 4. 7H 2 O, 0.05% KCl, 0.02% glucose and 0.02% sucrose) with or without 0.03 p m Myo5‐8

Article Snippet: Fusarium strains were grown on PDA for 3 days, and total RNA was extracted from the mycelia with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and was transcribed into cDNA as described previously (Song et al., ).

Techniques:

Journal: Molecular Plant Pathology

Article Title: Secondary amplification of siRNA machinery limits the application of spray‐induced gene silencing

doi: 10.1111/mpp.12728

Figure Lengend Snippet: The effects of Fa Myo5 double‐stranded RNA (dsRNA) on morphology and myosin‐5 gene expression of the different Fusarium species and other fungi. (A) Multiple sequence alignment of Fa Myo5 with its homologues from F. graminearum ,

Article Snippet: Fusarium strains were grown on PDA for 3 days, and total RNA was extracted from the mycelia with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and was transcribed into cDNA as described previously (Song et al., ).

Techniques: Expressing, Sequencing

Journal: Molecular Plant Pathology

Article Title: Secondary amplification of siRNA machinery limits the application of spray‐induced gene silencing

doi: 10.1111/mpp.12728

Figure Lengend Snippet: Secondary small interfering RNA (siRNA) amplification in Fusarium asiaticum .(A) Profiling of Myo5‐8 double‐stranded RNA (dsRNA)‐derived small RNAs (sRNAs) after Myo5‐8 dsRNA removal. Top panel is a diagram of eight fragments

Article Snippet: Fusarium strains were grown on PDA for 3 days, and total RNA was extracted from the mycelia with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and was transcribed into cDNA as described previously (Song et al., ).

Techniques: Small Interfering RNA, Amplification, Derivative Assay

Journal: Molecular Plant Pathology

Article Title: Secondary amplification of siRNA machinery limits the application of spray‐induced gene silencing

doi: 10.1111/mpp.12728

Figure Lengend Snippet: Double‐stranded RNA (dsRNA) uptake by the plant and effects on the fungal virulence of different pathways of Fusarium asiaticum uptake of dsRNA. (A) Microscopy of fluorescein‐Myo5‐8 dsRNA uptake by intact coleoptile and tip cut

Article Snippet: Fusarium strains were grown on PDA for 3 days, and total RNA was extracted from the mycelia with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and was transcribed into cDNA as described previously (Song et al., ).

Techniques: Microscopy

Journal: Molecular Plant Pathology

Article Title: Secondary amplification of siRNA machinery limits the application of spray‐induced gene silencing

doi: 10.1111/mpp.12728

Figure Lengend Snippet: Sensitivity of wild‐type (WT) and Myo5RNAi [ Myo5 gene RNA interference (RNAi) transformants] to osmotic and cell wall stress. (A) Growth of different strains on potato dextrose agar (PDA) plates or PDA amended with various compounds that generate

Article Snippet: Fusarium strains were grown on PDA for 3 days, and total RNA was extracted from the mycelia with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and was transcribed into cDNA as described previously (Song et al., ).

Techniques:

Journal: Nucleic Acids Research

Article Title: An antibody-based microarray assay for small RNA detection

doi: 10.1093/nar/gkl142

Figure Lengend Snippet: Comparison of antibody and cDNA methods for RNA detection. Spotted microarrays hybridized with 8 µg total E.coli RNA (Ambion) and detected by the monoclonal antibody method (top block) and cDNA method (lower block). Note that for the antibody method, signals are expected for the anti-sense spots (AS), while for the cDNA method, signals are expected for the sense spots (S). The grid at the bottom identifies the oligonucleotide in each position, with the naming conventions and sequences as shown in Supplementary Table S1. Quantification of fluorescent signals from two such arrays is presented in Table 1 .

Article Snippet: Total RNA Total E.coli RNA was purchased from Ambion (made from DH5α cultures harvested during the log phase of growth at an A 600 of 0.8, catalog no. 7940, Austin, TX) or isolated from exponentially-growing cultures of MG1655 (A 600 of 0.4) left untreated or exposed to 0.2 mM hydrogen peroxide for 5 min or overnight cultures of MC4100 cells using the hot-phenol extraction method as described previously ( ).

Techniques: RNA Detection, Blocking Assay

Journal: The Journal of Experimental Medicine

Article Title: Chemokine receptor CCR5 promotes leukocyte trafficking to the brain and survival in West Nile virus infection

doi: 10.1084/jem.20042530

Figure Lengend Snippet: West Nile virus induces production of specific immunoregulatory factors in the brains of C57BL/6 mice. (A) RNA analysis by RPA. Leftmost lane in each of the four panels is unprotected probe, and the factors analyzed are named to the left; note that the protected fragments run slightly lower on gel. The names of factors scoring positive in the assay are given to the right of each band. GAPDH is provided as an RNA loading control. Time after infection (days), is indicated at the top of each panel. Mock-infected mice were analyzed on day 7 of the experiment. Each lane on each panel corresponds to RNA from a single mouse brain. The results shown are from two independent experiments with six mice analyzed for each time point, except for mock-infected mice ( n = 3). Lanes corresponding in location on each of the four gels represent RNA from the same mouse. (B) RNA analysis by RT-PCR. β-Actin is provided as a loading control. Mock-infected mice were analyzed at day 7. Each lane represents a single mouse. (C) Protein analysis. The factor analyzed is identified at the top left of each graph. Time and virus variables for each factor are specified at the bottom of the panel. Data were pooled from three experiments with three to five in each group, and are presented as mean ± SEM pg/g protein in supernatants from total brain homogenates as a function of time after infection.

Article Snippet: RT-PCR Total RNA was extracted from homogenized brain tissue using TRIzol (Invitrogen).

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

Journal: Immunology

Article Title: Lactobacillus gasseri OLL2809 and its RNA suppress proliferation of CD4+ T cells through a MyD88-dependent signalling pathway

doi: 10.1111/j.1365-2567.2011.03455.x

Figure Lengend Snippet: LG2809 and its RNA suppressed proliferation of CD4 + T cells stimulated with anti-CD3/CD28 treatment in the absence of antigen-presenting cells. Plates were first coated with 10 μg/ml anti-CD3ε monoclonal antibody (mAb) for 2 hr at 37°. Subsequently, splenic CD4 + T cells from BALB/c mice (5 × 10 4 cells/well) and 2 μg/ml anti-CD28 mAb were added with indicated doses of heat-killed LG2809 (a), LG2809 RNA (b) or RNA from murine splenic cells (c) and were cultured for 72 hr. The cells were pulsed with [ 3 H]thymidine for 24 hr, and [ 3 H]thymidine incorporation was measured. Data are shown as the mean ± SD. Data are representative of three independent experiments. Statistical differences were analysed using Tukey's tests. Data are statistically different ( P

Article Snippet: Interestingly, E. coli Total RNA (Ambion) exhibited a suppressive effect on CD4+ T-cell proliferation, whereas RNA from mouse spleen Total RNA (Clontech, Palo Alto, CA) did not.

Techniques: Mouse Assay, Cell Culture

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Endotoxin Tolerant Dendritic Cells Suppress Inflammatory Responses in Splenocytes via Interleukin-1 Receptor Associated Kinase (IRAK)-M and Programmed Death-Ligand 1 (PDL-1)

doi: 10.12659/MSM.908242

Figure Lengend Snippet: Expression of IRAK-M and PDL-1 before and after the IRAK-M and PDL-1 inhibition of BMDCs. ( A, C ) Quantitative PCR analysis and western blot of IRAK-M expression of LPS primed or challenged DCs (n=3). ( B ) The expression of PDL-1 in LPS primed or challenged DCs (n=3). ( D ) IRAK-M mRNA and ( E ) protein expression after the interference using siRNA or si-RNA-controls to IRAK-M (n=3). ( F ) PDL-1 concentration of ELISA after using anti-PDL-1 antibody to neutralize supernatant PDL-1 (n=3). The expression of the error bars represents ±S.D. (* P

Article Snippet: Real-time RT-PCR Cell total RNA was extracted using Total RNA Purification Kit (Sigma, USA) and then reverse transcribed.

Techniques: Expressing, Inhibition, Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: BMC Plant Biology

Article Title: Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae

doi: 10.1186/s12870-015-0580-8

Figure Lengend Snippet: TRDMT1 and TRM4B methylate Arabidopsis nuclear encoded transfer RNAs. a Genomic origins of methylated and non-methylated tRNAs. Methylated tRNAs were only detected from the nuclear genome (3 biological replicates). b Above: clover-leaf representative secondary structure of tRNA indicating in red, the five cytosine positions methylated in wild type. Below: Heatmap showing percentage methylation of all cytosines detected in nuclear tRNAs of wild type, and mutants trdmt1 , trm4a , trm4b-1 and trdmt1 trm4b using RBS-seq. Cytosine positions are indicated next to tRNA isodecoders. White boxes represent cytosine positions with coverage less than five reads. (wild type 3 biological replicates, mutants n = 1). c Genomic structure of trm4a and trm4b mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by TRDMT1 at position C38 on BS treated tRNA Asp(GTC) template. Above: Restriction maps of PCR amplified products showing the expected digest patterns of methylated and non-methylated template. Below: Cleavage of PCR amplified product by HpyCH4IV confirms C38 methylation in wild type as opposed to non-methylated C38 in trdmt1 results in loss of HpyCH4IV restriction site. Loading control is undigested PCR product. e Hygromycin B stress assay. Trdmt1 trm4b double mutants and to a lesser extent, trm4b-1 mutants display increased sensitivity to hygromycin B (Hyg) at 10 and 20 days after germination (DAG) compared to controls

Article Snippet: To identify transcribed tRNAs, we initially used total RNA to construct an Illumina library, deep-sequenced the library and aligned the sequenced reads to our tRNA consensus list.

Techniques: Methylation, Polymerase Chain Reaction, Amplification

Journal: BMC Plant Biology

Article Title: Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae

doi: 10.1186/s12870-015-0580-8

Figure Lengend Snippet: Efficient detection of Arabidopsis tRNAs by polyacrylamide gel purification and RNA-seq. a Comparison of Illumina sequencing reads from either total RNA or gel purified RNA shows an increase in reads mapping to tRNAs from 0.0007 to 13.58 %, respectively. Data from one representative biological replicate is shown. b Venn diagram showing detection of gel purified tRNA consensus sequences from nuclear, chloroplast and mitochondrial genomes. 56 out of 100 known tRNA consensus sequences were identified in our analysis. Overlapping circles indicate tRNAs that may originate from more than one genome ( n = 3 biological replicates). c Consensus tRNAs display a wide range of expression levels with chloroplast (C) encoded sequences showing the highest expression levels compared to nuclear (N) and mitochondrial (M) sequences (1 replicate). Three of the tRNAs have undetermined anticodon sequences and are shown as (XXX). Minority isodecoders with diverged sequences from the majority isodecoder are designated by the number 1 or 2 after the anticodon. RBS-seq was used for ( a ) and ( b ) and RNA-seq was used in ( c )

Article Snippet: To identify transcribed tRNAs, we initially used total RNA to construct an Illumina library, deep-sequenced the library and aligned the sequenced reads to our tRNA consensus list.

Techniques: Gel Purification, RNA Sequencing Assay, Sequencing, Purification, Expressing

Journal: Scientific Reports

Article Title: Efficient Enrichment of Bacterial mRNA from Host-Bacteria Total RNA Samples

doi: 10.1038/srep34850

Figure Lengend Snippet: Depletion of Drosophila rRNA and actin transcripts. Coverage was compared for the Drosophila rRNA (panel A) and the actin gene (panel B) for the total (blue), polyA-selected (pink), and the bacterial mRNA-enriched (gray) RNA samples after normalizing for the number of reads sequenced, as calculated as NCPM, or n ormalized c overage p er m illion reads sequenced. rRNA is highly abundant in the total RNA, but significantly reduced in the polyA-selected and the bacterial mRNA-enriched samples. In contrast, the actin transcript was enriched only in the polyA-enriched sample. Therefore the method of bacterial mRNA enrichment was effective at removing both eukaryotic mRNA and rRNA.

Article Snippet: Bacterial mRNA Enrichment Using the Low Input Method Using 100 ng total RNA and a protocol distributed by Clontech ( http://www.clontech.com/JP/Products/cDNA_Synthesis_and_Library_Construction/Next_Gen_Sequencing_Kits/ibcGetAttachment.jsp?cItemId=75952 & fileId=6660677 & sitex=10025:22372:US ), the Ribo-Zero rRNA removal protocol (Epicentre, Madison, WI, USA) was carried out using a smaller amount of rRNA removal beads (90 μL), followed by the Invitrogen Dynabeads polyA enrichment protocol (Life Technologies, Grand Island, NY, USA) keeping the polyA-depleted material in the supernatant and discarding the poly-enriched material.

Techniques:

Journal: Scientific Reports

Article Title: Efficient Enrichment of Bacterial mRNA from Host-Bacteria Total RNA Samples

doi: 10.1038/srep34850

Figure Lengend Snippet: Bioanalyzer analysis of total RNA and bacterial mRNA-enriched samples from Drosophila ananassae colonized by its Wolbachia endosymbiont. The subtraction of Drosophila rRNA was assessed by running equivalent amounts of total RNA ( blue ) and Ribo-Zero reduced RNA ( pink ) on a Bioanalyzer. The software calculated the concentration of each sample by integrating the area under the rRNA peaks. Total RNA was 331 ng/μL and Ribo-Zero reduced RNA was 8 ng/μL, for an RNA loss of > 97%, most of which is in the rRNA peaks for both the bacterial endosymbiont and invertebrate host.

Article Snippet: Bacterial mRNA Enrichment Using the Low Input Method Using 100 ng total RNA and a protocol distributed by Clontech ( http://www.clontech.com/JP/Products/cDNA_Synthesis_and_Library_Construction/Next_Gen_Sequencing_Kits/ibcGetAttachment.jsp?cItemId=75952 & fileId=6660677 & sitex=10025:22372:US ), the Ribo-Zero rRNA removal protocol (Epicentre, Madison, WI, USA) was carried out using a smaller amount of rRNA removal beads (90 μL), followed by the Invitrogen Dynabeads polyA enrichment protocol (Life Technologies, Grand Island, NY, USA) keeping the polyA-depleted material in the supernatant and discarding the poly-enriched material.

Techniques: Software, Concentration Assay

Journal: Scientific Reports

Article Title: Efficient Enrichment of Bacterial mRNA from Host-Bacteria Total RNA Samples

doi: 10.1038/srep34850

Figure Lengend Snippet: Depletion of Wolbachia rRNA and enrichment of Wolbachia mRNA. Coverage was compared for the Wolbachia rRNA (panel A) and the WRi_010910 gene (panel B) for the total (blue), polyA-selected (pink), and bacterial mRNA-enriched (gray) samples after normalizing for the number of reads sequenced, as calculated as NCPB, or normalized coverage per billion reads sequenced. rRNA was highly abundant in the total RNA, but significantly reduced in the polyA-selected and the bacterial mRNA-enriched samples. In contrast, the WRi_010910 transcript was enriched in the bacterial mRNA-enriched sample compared to the total RNA. Therefore the method was effective at enriching for bacterial mRNA.

Article Snippet: Bacterial mRNA Enrichment Using the Low Input Method Using 100 ng total RNA and a protocol distributed by Clontech ( http://www.clontech.com/JP/Products/cDNA_Synthesis_and_Library_Construction/Next_Gen_Sequencing_Kits/ibcGetAttachment.jsp?cItemId=75952 & fileId=6660677 & sitex=10025:22372:US ), the Ribo-Zero rRNA removal protocol (Epicentre, Madison, WI, USA) was carried out using a smaller amount of rRNA removal beads (90 μL), followed by the Invitrogen Dynabeads polyA enrichment protocol (Life Technologies, Grand Island, NY, USA) keeping the polyA-depleted material in the supernatant and discarding the poly-enriched material.

Techniques:

Journal: Scientific Reports

Article Title: Efficient Enrichment of Bacterial mRNA from Host-Bacteria Total RNA Samples

doi: 10.1038/srep34850

Figure Lengend Snippet: Ehrlichia transcriptome sequencing coverage across a genome segment. The bacterial mRNA-enriched transcriptome sequencing coverage was plotted for the first 20 kbp of the Wakulla genome (Panel A) and compared to the predicted genes for this region (Panel B). While 99% of the genome is transcribed, troughs are apparent for tRNAs, which are too small to be recovered with the RNA isolation method used here. The coverage peaks at the 5′-end of transcripts and decays over the length of the transcript, as has been observed for other bacterial transcriptomes. Troughs are seen at transcriptional start sites, but not always at the 3′-ends of transcripts. This suggests that either the 3′-end of the transcripts overlap the ends of other transcripts, or that this strain lacks discrete transcription termination sites.

Article Snippet: Bacterial mRNA Enrichment Using the Low Input Method Using 100 ng total RNA and a protocol distributed by Clontech ( http://www.clontech.com/JP/Products/cDNA_Synthesis_and_Library_Construction/Next_Gen_Sequencing_Kits/ibcGetAttachment.jsp?cItemId=75952 & fileId=6660677 & sitex=10025:22372:US ), the Ribo-Zero rRNA removal protocol (Epicentre, Madison, WI, USA) was carried out using a smaller amount of rRNA removal beads (90 μL), followed by the Invitrogen Dynabeads polyA enrichment protocol (Life Technologies, Grand Island, NY, USA) keeping the polyA-depleted material in the supernatant and discarding the poly-enriched material.

Techniques: Sequencing, Isolation

Journal: Nature

Article Title: Subepithelial telocytes are an important source of Wnts that supports intestinal crypts

doi: 10.1038/s41586-018-0084-4

Figure Lengend Snippet: Foxl1 + telocytes provide essential Wnt ligands to the intestinal stem cell compartment (a,b) Wnt pathway activation analyzed by immunohistochemistry for β catenin (brown). Insets a′ and b′ at high magnification. (c–j) Immunofluorescence staining of the Wnt targets CyclinD1 and Sox9. (k–n) Stem cell markers Olfm4 (in situ hybridization) and CD44 (immunohistochemistry) one day post induction in PorcnΔ duodenum. Insets k′, l′, m′, n′ represent high magnification. (o,p) Expression of Lgr5 and Rspo3 as detected by single molecule RNA-FISH three day post induction in PorcnΔ duodenum. * denotes a staining artifact. (q,r) Immunofluorescence for EpCAM and PDGRFα in duodenum of control and PorcnΔ mice. Experiments were repeated for at least three times with similar results. Scale bars 100 μm (a–n) and 25 μm in insets

Article Snippet: For RNA isolation, GFP+ and Tomato+ cells for Foxl1+ and Foxl1− mesenchymal cells, respectively, were lysed and total RNA was isolated by column purification (Absolutely RNA Nanoprep Kit; Agilent Technologies). cDNA was synthesized and amplified using Ribo-SPIA technology (Ovation RNA-Seq System V2 #7102 NuGEN). cDNA was sonicated and an mRNA sequencing library was prepared using the NEBNext RNA library prep kit (New England BioLabs, Inc).

Techniques: Activation Assay, Immunohistochemistry, Immunofluorescence, Staining, In Situ Hybridization, Expressing, Fluorescence In Situ Hybridization, Mouse Assay

Journal: Free radical biology & medicine

Article Title: NRF2 Regulates Hyperoxia-induced NOX4 expression in Human Lung Endothelium: Identification of functional antioxidant response elements on NOX4 promoter

doi: 10.1016/j.freeradbiomed.2011.03.022

Figure Lengend Snippet: NRF2 siRNA increases hyperoxia-induced ROS production in HLMVECs Panel A confirms reduction in NRF2 protein expression by NRF2 siRNA. HLMVECs grown on 35-mm dishes to ~ 50% confluence were transfected with scrambled siRNA (Sc) or NRF2 siRNA (50nM, 48 h). Cell lysates (30 μg proteins) were extracted and subjected to SDS-PAGE, and analyzed by Western blotting with anti-NRF2 antibody as described shown under “Materials and Methods”. Shown is a representative blot from three independent experiments. In Panel B , HLMVECs grown on 35-mm dishes to ~ 50% confluence were transfected with scrambled siRNA or NRF2 siRNA (50nM) for 48 h, total RNA was extracted and NOX4 mRNA expression was quantified by real time RT-PCR and normalized to 18S rRNA. Values are average of three independent determinations. *, significantly different from scrambled siRNA transfected cells ( P

Article Snippet: Total RNA was isolated from HLMVECs grown on 35-mm dishes using TRIzol® reagent according to the manufacturer’s instruction. iQ SYBR Green Supermix was used to do the real time using iCycler by Biorad.

Techniques: Expressing, Transfection, SDS Page, Western Blot, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Short-lived AUF1 p42-binding mRNAs of RANKL and BCL6 have two distinct instability elements each

doi: 10.1371/journal.pone.0206823

Figure Lengend Snippet: Binding-sites for AUF1 p37, HuR and KSRP in the mouse RANKL 3’UTR. Various pZPCTHI constructs with long or short inserts of the 3’UTR of mouse RANKL mRNA were transiently transfected with Lipofectamine 2000 into HEK 293T cells along with vectors to express tagged ARE-binding proteins. 21 h post transfection, cells were lysed and protein-bound RNA co-immunoprecipitated and recovered as shown in Fig 1 and described in Materials and Methods. RNA was isolated from input, microbead-adsorbed pellet and non-bound supernatant fractions, and EGFP and endogenous GAPDH mRNA quantified by RT-PCR. For each recombinant ARE-binding protein, the % adsorbed versus totally recovered RNA was calculated, and the relative enrichment of EGFP mRNA versus GAPDH mRNA expressed as “n-fold enrichment” ± standard deviation (number independent experiments). The graphs below show a graphical representation of the results. Vertical black bars indicate the location of instability elements.

Article Snippet: cRNA synthesis and microarray hybridization For the analysis on Affymetrix GeneChips (Affymetrix, Santa Clara, CA) 50 ng of immunoprecipitated RNA or of a total RNA control sample were processed with the two-cycle target labelling protocol (Technical Manual; Gene Expression Analysis 701021 Rv.3 of Affymetrix) to produce biotinylated cRNA target.

Techniques: Binding Assay, Construct, Transfection, Immunoprecipitation, Isolation, Reverse Transcription Polymerase Chain Reaction, Recombinant, Standard Deviation

Journal: PLoS ONE

Article Title: Short-lived AUF1 p42-binding mRNAs of RANKL and BCL6 have two distinct instability elements each

doi: 10.1371/journal.pone.0206823

Figure Lengend Snippet: Binding-sites for AUF1 p37, HuR and KSRP in the human BCL6 3’UTR. Various pZPCTHI constructs with long or short inserts of the 3’UTR of human BCL6 mRNA were transiently transfected with Lipofectamine 2000 into HEK 293T cells along with vectors to express tagged ARE-binding proteins. 21 h post transfection, cells were lysed and protein-bound RNA co-immunoprecipitated and recovered as shown in Fig 1 and described in Materials and Methods. RNA was isolated from input, microbead-adsorbed pellet and non-bound supernatant fractions, and EGFP and endogenous GAPDH mRNA quantified by RT-PCR. For each recombinant ARE-binding protein, the % adsorbed versus totally recovered RNA was calculated, and the relative enrichment of EGFP mRNA versus GAPDH mRNA expressed as “n-fold enrichment” ± standard deviation (number independent experiments). The graphs below show a graphical representation of the results. Vertical black bars indicate the location of instability elements.

Article Snippet: cRNA synthesis and microarray hybridization For the analysis on Affymetrix GeneChips (Affymetrix, Santa Clara, CA) 50 ng of immunoprecipitated RNA or of a total RNA control sample were processed with the two-cycle target labelling protocol (Technical Manual; Gene Expression Analysis 701021 Rv.3 of Affymetrix) to produce biotinylated cRNA target.

Techniques: Binding Assay, Construct, Transfection, Immunoprecipitation, Isolation, Reverse Transcription Polymerase Chain Reaction, Recombinant, Standard Deviation

Journal: Iranian Biomedical Journal

Article Title: Design and Development of Modified mRNA Encoding Core Antigen of Hepatitis C Virus: a Possible Application in Vaccine Production

doi: 10.29252/.23.1.57

Figure Lengend Snippet: PCR products of cloned sequences and mRNA transcript. (A) The amplified fragments obtained from the enzymatic digestion of plasmids carrying Core , SP- Core , and GFP genes ; (B) The amplified HCV core mRNA transcript (1.6 kb) running on an agarose gel (1.1%). M, RNA marker

Article Snippet: To remove the template DNA, 1 μl of RQ1 RNase-Free DNase (Catalog Numbers: M6101) was added to the IVT reaction mixture and incubated at 37°C for 15 min. Purification of mRNA was performed after DNaseI digestion by Total RNA Purification Kit (Jena-Bioscience , Germany), according to the instructions provided by manufacturer.

Techniques: Polymerase Chain Reaction, Clone Assay, Amplification, Agarose Gel Electrophoresis, Marker

Journal: Nature

Article Title: A long non-coding RNA protects the heart from pathological hypertrophy

doi: 10.1038/nature13596

Figure Lengend Snippet: Mhrt inhibits cardiac hypertrophy and failure a, Quantitation of cardiac Mhrt s 2–42 days after TAC operation. P-value: Student’s t-test. Error bar: SEM. b, RT-PCR of Mhrts in adult heart ventricles. Primers (F1 and R1, Fig. 1a ) target Mhrt common regions. Size controls 779, 826, 709 are PCR products of recombinant Mhrt779, Mhrt826 , and Mhrt709 , respectively. c , Northern blot of Mhrts in adult heart ventricles. The probe targets common regions of Mhrts . Negative: control RNA from 293T cells. Size control 826 is recombinant Mhrt826 ; 643 (not a distinct Mhrt species) contains the 5′ common region of Mhrt . d Quantitation of Mhrt779 in control or Tg779 mice with/without doxycycline (Dox) or TAC operation. Mhrt779 -specific PCR primers were used. Ctrl: control mice. Tg779 : Tnnt2-rtTA ; Tre-Mhrt779 mice. P-value: Student’s t-test. Error bar: standard error of the mean (SEM). e, Ventricle-body weight ratio of hearts 6 weeks after TAC. P-value: Student’s t-test. Error bar: SEM. Scale=1 mm. f, Quantitation of cardiomyocyte size in control and Tg779 mice 6 weeks after TAC by wheat-germ agglutinin staining. P-value: Student’s t-test. Error bar: SEM. g, Trichrome staining in control and Tg779 hearts 6 weeks after TAC. Red: cardiomyocytes. Blue: fibrosis. h, i, Echocardiographic measurement of left ventricular fractional shortening ( h ) and internal dimensions at end-diastole (LVIDd) and end-systole (LVIDs) ( i ) 6 weeks after TAC. P-value: Student’s t-test. Error bar: SEM.

Article Snippet: To conduct strand specific RT PCR analysis, human total RNA and Superscript III First-Strand Synthesis System (Invitrogen) was used.

Techniques: Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Recombinant, Northern Blot, Negative Control, Mouse Assay, Staining

Journal: Nature

Article Title: A long non-coding RNA protects the heart from pathological hypertrophy

doi: 10.1038/nature13596

Figure Lengend Snippet: Profile of the non-coding RNA Mhrt a, Schematic illustration of Mhrt s originating from the intergenic region between Myh6 and Myh7 and transcribed into Myh7. Myh7 exons and introns are indicated. F1 and R1, targeting 5' and 3′ Mhrt common sequences, are the primers used for subsequent PCR. b, RT-qPCR of Mhrts using primers targeting common regions of Mhrts in tissues from 2-month-old mice. P-value: Student’s t-test. Error bar: standard error of the mean (SEM). c, RT-qPCR of Mhrt, Myh6 and Myh7 in mouse hearts at different ages. Mhrt and Myh6 / Myh7 ratio of E11 hearts are set as 1. Error bar: SEM. d, RNA in situ analysis of Mhrt (blue) in adult hearts. The RNA probe targets all Mhrt species. Red: nuclear fast red. White arrowheads: myocardial nuclei. Black arrowheads: nuclei of endothelial, endocardial or epicardial cells. Dashed lines demarcate the myocardium from endocardium (endo) or from epicardium (epi). Scale= 50 μm. e, RT-qPCR of nuclear/cytoplasmic RNA in adult hearts. TfIIb, Hprt1, and 28SrRNA are primarily cytoplasmic RNAs; Neat1, nuclear lncRNA. TfIIb ratio is set as 1. P-value: Student’s t-test. Error bar: SEM. f, Ribosome profiling: ribosome density on coding RNAs and lncRNAs.

Article Snippet: To conduct strand specific RT PCR analysis, human total RNA and Superscript III First-Strand Synthesis System (Invitrogen) was used.

Techniques: Polymerase Chain Reaction, Quantitative RT-PCR, Mouse Assay, In Situ

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

Article Title: Intron retention induced by microsatellite expansions as a disease biomarker

doi: 10.1073/pnas.1716617115

Figure Lengend Snippet: CNBP intron 1 retention in DM2 as a blood biomarker. ( A ) UCSC browser view of CNBP and wiggle plots of PBL RNA-seq data from DM2 ( n = 3) and ALS ( n = 5) controls. ( B ). Two-tailed t test: ** P = 0.0018. ( C ) Southern blot analysis of genomic DNA derived from DM2 patient PBLs with small (100–400 CCTGs) and large (≥1,000 CCTGs) expansions with DM1 disease and unaffected controls (Ctrl.). ( D ) CNBP i1 3′ss RT-PCR analysis of PBLs from DM2 patients with large ( n = 5) and small ( n = 4) CCTG expansions, DM1 ( n = 2), ALS ( n = 9), and unaffected controls ( n = 2). ( E ) Bar graph shows mean ± SD for CNBP i1 retention ratio. One-way ANOVA with Tukey’s multiple comparison test: * P = 0.0135; ** P = 0.0055; *** P

Article Snippet: PBLs were centrifuged, washed once with PBS, and used for either gDNA isolation (Flexigene kit; Qiagen), LCL generation, or total RNA isolation (TRIzol; Thermo Fisher Scientific) per the manufacturer’s protocols.

Techniques: Biomarker Assay, RNA Sequencing Assay, Two Tailed Test, Southern Blot, Derivative Assay, Reverse Transcription Polymerase Chain Reaction

Journal: BMC Veterinary Research

Article Title: Curcumin is a promising inhibitor of genotype 2 porcine reproductive and respiratory syndrome virus infection

doi: 10.1186/s12917-017-1218-x

Figure Lengend Snippet: Evaluation of the effect of curcumin on PRRSV entry and levels of receptors in PAMs. Treatment of PAMs was performed as in Fig. 1 . After incubation for 36 h, total cellular RNA was extracted and PRRSV ORF7 RNA levels were detected by qRT-PCR ( a ). Culture supernatants were tested for virus titers ( b ). Curcumin cytotoxicity was detected in PAMs using a CCK8 assay and is presented as experimental cell viability relative to cell viability of untreated control (100%) ( c ). PAMs were incubated with curcumin (15 μM) at 37 °C for 1 h and CD163, MYH9, vimentin, and CD169 protein levels were detected by western blot. In addition, PAMs were incubated with curcumin at 37 °C for 1 h and then the cells were infected with PRRSV GD-HD strain (MOI: 0.1). After 36 h, PRRSV N protein expression levels were determined by western blot ( d ). Each value represents the mean ± SD from three independent experiments (*, p

Article Snippet: Quantitative reverse transcription-PCR (qRT-PCR) Total RNA was extracted from Marc-145 cells using Total RNA Kit (OMEGA Bio-Tek, GA, USA) using the manufacturer’s instructions.

Techniques: Incubation, Quantitative RT-PCR, CCK-8 Assay, Western Blot, Infection, Expressing

Journal: BMC Veterinary Research

Article Title: Curcumin is a promising inhibitor of genotype 2 porcine reproductive and respiratory syndrome virus infection

doi: 10.1186/s12917-017-1218-x

Figure Lengend Snippet: Measurement of a curcumin effect on PRRSV absorption in Marc-145 cells. Marc-145 cells were pre-chilled at 4 °C for 1 h followed by incubation with curcumin-pretreated GD-HD (MOI: 100) ( a ), or a mixture of GD-HD (MOI: 100) and curcumin ( c ) at 4 °C for 1 h. For pretreatment of cells, Marc-145 cells were pre-incubated with curcumin at 37 °C for 1 h. After incubating cells at 4 °C for 1 h, both GD-HD (MOI: 100) and curcumin were added and incubated for 1 h at 4 °C ( b ). After washing out unbound virus, total RNA was extracted from the cells and the relative cell-bound PRRSV RNA levels were measured using qRT-PCR

Article Snippet: Quantitative reverse transcription-PCR (qRT-PCR) Total RNA was extracted from Marc-145 cells using Total RNA Kit (OMEGA Bio-Tek, GA, USA) using the manufacturer’s instructions.

Techniques: Incubation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: GmCYP82A3, a Soybean Cytochrome P450 Family Gene Involved in the Jasmonic Acid and Ethylene Signaling Pathway, Enhances Plant Resistance to Biotic and Abiotic Stresses

doi: 10.1371/journal.pone.0162253

Figure Lengend Snippet: Expression profiles of defense marker genes in transgenic N . benthamiana plants. Total RNA was extracted from detached leaves of WT, EV and GmCYP82A3 overexpression plants at 0, 12, 24 hpi by P . parasitica zoospores. Expression levels were determined by qRT-PCR using gene specific primers and normalized to NbEF1a with three replicate experiments. Data are the means of three replications, error bars indicate SD. The significant differences between WT and transgenic plants are indicated by asterisk (Dunnett-t test, * P

Article Snippet: RNA isolation, semi-quantitative and quantitative real-time PCR Total RNA was extracted using the Total RNA kit (Tiangen, CHINA), gDNA elimination and reverse transcription were performed with the PrimeScript™ RT reagent kit (TaKaRa, JAPAN).

Techniques: Expressing, Marker, Transgenic Assay, Over Expression, Quantitative RT-PCR