Journal: Biology Open

Article Title: Star-PAP regulates tumor protein D52 through modulating miR-449a/34a in breast cancer

doi: 10.1242/bio.045914

Figure Lengend Snippet: Star-PAP regulated miR-449a/34a expressions. (A) Overexpression of Star-PAP for 24 h. The expression of miR-449a/34a was detected by qPCR. (B) Decreased levels of miR-449a/34a following Star-PAP knockdown by siRNAs for 48 h. (C) Cells were co-transfected with either pFlagcmv2-Star-PAP and 80 ng plasmid carrying either WT or Mut 3′-UTR of TPD52. The relative firefly luciferase activity normalized with Renilla luciferase was measured 24 h after transfection. (D) RNA pull down assay was performed by 5′-biotin-miR-449a and biotin-Scramble. 293 FT cells were transfected with pFlagcmv2-Star-PAP for 24 h. Cells were lysed in RIPA buffer, then incubated with biotin-miR-449a or biotin-scramble for 4 h, before that they were pre-incubated with miR-449a or Scramble for 1 h, followed by adding avidin beads, and finally examined by western blot. (E) Cells were co-transfected with Star-PAP and miR-449a mimic or inhibitor, the TPD52 mRNA level was detected by qPCR. (F) The same treatment as with E, and the protein levels were examined. (G) Quantification of TPD52 protein level in F by NIH ImageJ software. Data are means±s.d. ( n =3) with three independent repeats, * P

Article Snippet: For detection of mature miR-449a/34a, total RNA was subjected to reverse TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems), and TaqMan MicroRNA Assay Kit (Applied Biosystems) was used for qPCR analysis.

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Pull Down Assay, Incubation, Avidin-Biotin Assay, Western Blot, Software

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: 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: 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: International Journal of Molecular Medicine

Article Title: Anti-atherogenic effect of Humulus japonicus in apolipoprotein E-deficient mice

doi: 10.3892/ijmm.2016.2727

Figure Lengend Snippet: Effect of H. japonicus (HJ) on pro-atherogenic gene expression in the aorta of apolipoprotein E-deficient (apoE −/− ) mice. The apoE −/− mice were fed an atherogenic diet plus either vehicle [0.5% carboxymethyl cellulose (CMC)] as the vehicle group (unfilled bar), and 500 mg/kg of HJ as the HJ500 group (filled bar) for 12 weeks. The whole aortas from 3–4 mice in each group were pooled to extract RNA. The mRNA expression of (A) vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), (B) inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), (C) monocyte chemoattractant protein-1 (MCP-1) and CD68, and (D) interleukin (IL)-1β, IL-6, and IL-18 as measured by RT-qPCR and normalized by 18s mRNA signal. The values are presented as the means ± SEM. Statistical significance relative to vehicle group, * P

Article Snippet: Assessment of the aortic expression of atherogenic genes in apoE−/− mice Total RNA from the whole aorta in each group was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions, quantified by a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, San Diego, CA, USA), and stored at −70°C until analysis.

Techniques: Expressing, Mouse Assay, Quantitative RT-PCR