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  • 99
    Thermo Fisher total rna
    Star-PAP regulated <t>miR-449a/34a</t> 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) <t>RNA</t> 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
    Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 478353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    TaKaRa total rnas
    Overall small <t>RNA</t> dynamics during rice grain filling processes. (A) . Line charts showing the length-distribution of total small <t>RNAs</t> from 10 deep sequenced libraries of both superior and inferior spikelets at different rice grain filling stages. (B) . Dot chart showing different percentages of the 21- and 24-nt total small RNAs presenting in superior and inferior spikelets at different rice grain filling stages. (C) . Bar graph showing the small RNA dynamics of different categories from the 10 libraries of different grain filling stages.
    Total Rnas, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 3141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher total rnas
    Tel-sRNA generation is correlated with genome reorganization and heterochromatin formation in Tetrahymena thermophila . (A) Northern blot analyses of <t>RNAs</t> collected at different times of conjugation (shown on top ) from wild-type cells. For RNase treatment, RNAs extracted at 4 h of conjugation were used. (B) Quantitative RT-PCR of tel-sRNAs using <t>RNA</t> samples in A . Signals were presented as fold differences relative to Tel-C signal in 0 h of conjugation, which was arbitrarily set as 1. (C) Northern blot analyses of RNAs collected at different time of conjugation (shown on top ) from or Dcl1 (−/−) cells. In both A and C , the blots were hybridized with (ACCCCA) 3 , (TGGGGT) 3 , or M-scnRNA probes, respectively. The transcripts were indicated on the right . Ethidium bromide stained PAGE gel was shown at bottom as the loading control.
    Total Rnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 23282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene total rnas
    Tel-sRNA generation is correlated with genome reorganization and heterochromatin formation in Tetrahymena thermophila . (A) Northern blot analyses of <t>RNAs</t> collected at different times of conjugation (shown on top ) from wild-type cells. For RNase treatment, RNAs extracted at 4 h of conjugation were used. (B) Quantitative RT-PCR of tel-sRNAs using <t>RNA</t> samples in A . Signals were presented as fold differences relative to Tel-C signal in 0 h of conjugation, which was arbitrarily set as 1. (C) Northern blot analyses of RNAs collected at different time of conjugation (shown on top ) from or Dcl1 (−/−) cells. In both A and C , the blots were hybridized with (ACCCCA) 3 , (TGGGGT) 3 , or M-scnRNA probes, respectively. The transcripts were indicated on the right . Ethidium bromide stained PAGE gel was shown at bottom as the loading control.
    Total Rnas, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 211 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher human total rna total rna
    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, <t>RT-PCR</t> 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 <t>RNA</t> 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.
    Human Total Rna Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher total rna control
    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 <t>RNA</t> <t>co-immunoprecipitated</t> 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.
    Total Rna Control, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega total rna total rna
    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 <t>RNA</t> <t>co-immunoprecipitated</t> 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.
    Total Rna Total Rna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher mice total rna
    Effect of H. japonicus (HJ) on pro-atherogenic gene expression in the aorta of apolipoprotein E-deficient <t>(apoE</t> −/− ) 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 <t>RNA.</t> 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
    Mice Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher total rna extraction total rna
    Effect of H. japonicus (HJ) on pro-atherogenic gene expression in the aorta of apolipoprotein E-deficient <t>(apoE</t> −/− ) 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 <t>RNA.</t> 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
    Total Rna Extraction Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1569 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher firstchoice total rna
    Effect of H. japonicus (HJ) on pro-atherogenic gene expression in the aorta of apolipoprotein E-deficient <t>(apoE</t> −/− ) 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 <t>RNA.</t> 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
    Firstchoice Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher pancreas total rna
    Effect of H. japonicus (HJ) on pro-atherogenic gene expression in the aorta of apolipoprotein E-deficient <t>(apoE</t> −/− ) 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 <t>RNA.</t> 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
    Pancreas Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa pancreas development total rna
    Effect of H. japonicus (HJ) on pro-atherogenic gene expression in the aorta of apolipoprotein E-deficient <t>(apoE</t> −/− ) 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 <t>RNA.</t> 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
    Pancreas Development Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Agilent technologies total rna
    <t>Foxl1</t> + 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 <t>RNA-FISH</t> 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
    Total Rna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 15580 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc total rna
    TRDMT1 and TRM4B methylate Arabidopsis nuclear encoded transfer RNAs. a Genomic origins of methylated and non-methylated <t>tRNAs.</t> 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 <t>RNA</t> 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
    Total Rna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 12657 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    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

    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 .

    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

    Detecting the expression levels of adhesion molecules. a Total RNA from whole cell lysates with different treatment, cDNA were synthesized according to the procedure of HiFiScript cDNA Synthesis Kit. RT-qPCR was used to detect the expression levels of HAS1, HAS2, HAS3, CD44 and OPN mRNA in NRK-52E cells exposed with COM (146.0 µg/cm 2 ), whereas GAPDH served as the loading control. N = 3 independent experiments for each bar; * P

    Journal: Urolithiasis

    Article Title: P38 MAPK signaling pathway mediates COM crystal-induced crystal adhesion change in rat renal tubular epithelial cells

    doi: 10.1007/s00240-019-01143-z

    Figure Lengend Snippet: Detecting the expression levels of adhesion molecules. a Total RNA from whole cell lysates with different treatment, cDNA were synthesized according to the procedure of HiFiScript cDNA Synthesis Kit. RT-qPCR was used to detect the expression levels of HAS1, HAS2, HAS3, CD44 and OPN mRNA in NRK-52E cells exposed with COM (146.0 µg/cm 2 ), whereas GAPDH served as the loading control. N = 3 independent experiments for each bar; * P

    Article Snippet: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis The total RNA from NRK-52E cells were extracted using the Trizol Reagent (Invitrogen, USA), and cDNA was then synthesized using a reverse transcription (RT) system kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions.

    Techniques: Expressing, Synthesized, Quantitative RT-PCR

    Ig gene rearrangement and transcription in μMT mice liver epithelial cells. ( A ) Northern blot analysis. Total RNA from liver cells of three μMT mice and WT spleen cells (positive control) were transferred to nylon membrane, and probed with probes to constant region of Igμ and Igκ; 28 s and 18 s RNA were shown by DNA agarose gel electrophoresis. ( B ) In situ hybridization analysis was shown for detection of Ig μ and Ig κ transcripts in μMT mice with antisense or sense probes for C region of μ chain or J-C region of κ chain. ( C ) a It was shown for the primer design of Ig μ variable region, Ig κ variable region and Igλ; b Sorted liver epithelial cells from two μMT mice were analyzed for Ig rearrangement and transcription by RT-PCR. WT spleen cells were used as the positive control. Variable region and constant region of heavy chain (μ, α, δ, γ, ε) and light chain (κ, λ), CD20 and GAPDH were amplified; “1, 2” represented two samples of sorted μMT liver epithelial cells.

    Journal: Scientific Reports

    Article Title: Identification of Liver Epithelial Cell-derived Ig Expression in μ chain-deficient mice

    doi: 10.1038/srep23669

    Figure Lengend Snippet: Ig gene rearrangement and transcription in μMT mice liver epithelial cells. ( A ) Northern blot analysis. Total RNA from liver cells of three μMT mice and WT spleen cells (positive control) were transferred to nylon membrane, and probed with probes to constant region of Igμ and Igκ; 28 s and 18 s RNA were shown by DNA agarose gel electrophoresis. ( B ) In situ hybridization analysis was shown for detection of Ig μ and Ig κ transcripts in μMT mice with antisense or sense probes for C region of μ chain or J-C region of κ chain. ( C ) a It was shown for the primer design of Ig μ variable region, Ig κ variable region and Igλ; b Sorted liver epithelial cells from two μMT mice were analyzed for Ig rearrangement and transcription by RT-PCR. WT spleen cells were used as the positive control. Variable region and constant region of heavy chain (μ, α, δ, γ, ε) and light chain (κ, λ), CD20 and GAPDH were amplified; “1, 2” represented two samples of sorted μMT liver epithelial cells.

    Article Snippet: RNA extraction and RT-PCR Total RNA in spleen was extracted using TRIzol reagent (Invitrogen, Carlsbad, USA).

    Techniques: Mouse Assay, Northern Blot, Positive Control, Agarose Gel Electrophoresis, In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Amplification

    Overall small RNA dynamics during rice grain filling processes. (A) . Line charts showing the length-distribution of total small RNAs from 10 deep sequenced libraries of both superior and inferior spikelets at different rice grain filling stages. (B) . Dot chart showing different percentages of the 21- and 24-nt total small RNAs presenting in superior and inferior spikelets at different rice grain filling stages. (C) . Bar graph showing the small RNA dynamics of different categories from the 10 libraries of different grain filling stages.

    Journal: BMC Plant Biology

    Article Title: Differentially expressed microRNA cohorts in seed development may contribute to poor grain filling of inferior spikelets in rice

    doi: 10.1186/s12870-014-0196-4

    Figure Lengend Snippet: Overall small RNA dynamics during rice grain filling processes. (A) . Line charts showing the length-distribution of total small RNAs from 10 deep sequenced libraries of both superior and inferior spikelets at different rice grain filling stages. (B) . Dot chart showing different percentages of the 21- and 24-nt total small RNAs presenting in superior and inferior spikelets at different rice grain filling stages. (C) . Bar graph showing the small RNA dynamics of different categories from the 10 libraries of different grain filling stages.

    Article Snippet: RNA ligase–mediated 5’-RACE To conduct the RNA ligase–mediated 5’-RACE, 1 μg total RNAs from equal mixture of superior and inferior spikelets at 10DAF, 15DAF, 21DAF, 27DAF, and 35DAF was ligated to a 5’-RACE RNA adapter without calf intestine alkaline phosphatase treatment using 5′-Full RACE Kit (Takara), followed by a reverse transcription reaction.

    Techniques:

    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.

    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:

    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.

    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

    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.

    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:

    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.

    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

    Tel-sRNA generation is correlated with genome reorganization and heterochromatin formation in Tetrahymena thermophila . (A) Northern blot analyses of RNAs collected at different times of conjugation (shown on top ) from wild-type cells. For RNase treatment, RNAs extracted at 4 h of conjugation were used. (B) Quantitative RT-PCR of tel-sRNAs using RNA samples in A . Signals were presented as fold differences relative to Tel-C signal in 0 h of conjugation, which was arbitrarily set as 1. (C) Northern blot analyses of RNAs collected at different time of conjugation (shown on top ) from or Dcl1 (−/−) cells. In both A and C , the blots were hybridized with (ACCCCA) 3 , (TGGGGT) 3 , or M-scnRNA probes, respectively. The transcripts were indicated on the right . Ethidium bromide stained PAGE gel was shown at bottom as the loading control.

    Journal: RNA

    Article Title: Dicer independent small RNAs associate with telomeric heterochromatin

    doi: 10.1261/rna.1423309

    Figure Lengend Snippet: Tel-sRNA generation is correlated with genome reorganization and heterochromatin formation in Tetrahymena thermophila . (A) Northern blot analyses of RNAs collected at different times of conjugation (shown on top ) from wild-type cells. For RNase treatment, RNAs extracted at 4 h of conjugation were used. (B) Quantitative RT-PCR of tel-sRNAs using RNA samples in A . Signals were presented as fold differences relative to Tel-C signal in 0 h of conjugation, which was arbitrarily set as 1. (C) Northern blot analyses of RNAs collected at different time of conjugation (shown on top ) from or Dcl1 (−/−) cells. In both A and C , the blots were hybridized with (ACCCCA) 3 , (TGGGGT) 3 , or M-scnRNA probes, respectively. The transcripts were indicated on the right . Ethidium bromide stained PAGE gel was shown at bottom as the loading control.

    Article Snippet: For small RNA RT-PCR analysis, total RNAs were treated with DNase I (Ambion) and subject to reverse transcription using a miScript reverse transcription kit (Qiagen).

    Techniques: Northern Blot, Conjugation Assay, Quantitative RT-PCR, Staining, Polyacrylamide Gel Electrophoresis

    Analysis of telomeric small RNA transcripts in mouse embryonic stem cells. (A) Total RNA was size fractionated using a PureLink miRNA isolation kit. Samples of small RNA molecules from wild-type and Dicer (−/−) cells were analyzed by Northern blot after DNase I treatment. A 32 P-end labeled (CCCTAA) 3 oligonucleotide probe was used to detect small-sized telomeric transcripts. For sizing control, the radio labeled RNA decade marker (Ambion) is shown on the left . The blot was reprobed with miR-293 ( middle panel) and tRNA-Ile-ATT, which serves as a loading control ( bottom panel). (B) The small RNAs treated with and without sodium periodate and β-elimination were separated on a 15% gel. The 32 P-end labeled (CCCTAA) 3 oligonucleotide probe was used to detect small-sized telomeric transcripts. RNA marker is shown on the left . As a control, a synthetic RNA oligo (with free 2′OH) treated with or without sodium periodate and β-elimination were 5′ end labeled with 32 P and separated on a 12% gel. (C) Quantitative PCR for tel-sRNAs in various cell lines as indicated on the bottom .

    Journal: RNA

    Article Title: Dicer independent small RNAs associate with telomeric heterochromatin

    doi: 10.1261/rna.1423309

    Figure Lengend Snippet: Analysis of telomeric small RNA transcripts in mouse embryonic stem cells. (A) Total RNA was size fractionated using a PureLink miRNA isolation kit. Samples of small RNA molecules from wild-type and Dicer (−/−) cells were analyzed by Northern blot after DNase I treatment. A 32 P-end labeled (CCCTAA) 3 oligonucleotide probe was used to detect small-sized telomeric transcripts. For sizing control, the radio labeled RNA decade marker (Ambion) is shown on the left . The blot was reprobed with miR-293 ( middle panel) and tRNA-Ile-ATT, which serves as a loading control ( bottom panel). (B) The small RNAs treated with and without sodium periodate and β-elimination were separated on a 15% gel. The 32 P-end labeled (CCCTAA) 3 oligonucleotide probe was used to detect small-sized telomeric transcripts. RNA marker is shown on the left . As a control, a synthetic RNA oligo (with free 2′OH) treated with or without sodium periodate and β-elimination were 5′ end labeled with 32 P and separated on a 12% gel. (C) Quantitative PCR for tel-sRNAs in various cell lines as indicated on the bottom .

    Article Snippet: For small RNA RT-PCR analysis, total RNAs were treated with DNase I (Ambion) and subject to reverse transcription using a miScript reverse transcription kit (Qiagen).

    Techniques: Isolation, Northern Blot, Labeling, Marker, Real-time Polymerase Chain Reaction

    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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

    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

    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

    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

    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

    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

    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 )

    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