Journal: Nature Communications

Article Title: Platelets reduce anoikis and promote metastasis by activating YAP1 signaling

doi: 10.1038/s41467-017-00411-z

Figure Lengend Snippet: YAP1 is activated by platelets and is indispensable for platelet-induced anoikis resistance. a , b Western blot analysis of phosphorylated YAP1 (S127 and S397) and total YAP1 in HEYA8 a and OVCAR8 b cells after two hours under low-attachment conditions with or without platelet co-incubation. GAPDH was used as a loading control ( n = 5). c , d Western blot analysis of phosphorylated YAP1 (S127) and total YAP1 in SKOV3, MDAH-2774, OVCAR4, SW620 c and OVCAR5, OVCA432 and RKO d GAPDH was used as a loading control ( n = 3). e , f Immunofluorescence staining of YAP1 in HEYA8 e and OVCAR8 f cells after two hours under low-attachment conditions with ( lower panels ) or without ( upper panels ) platelet co-incubation. Inlets showing higher magnification of cells on the right side of the panels. Nuclear counterstain was done using Hoechst 33342 ( n = 3). Scale bars = 20 µm. g QRT–PCR and western blot analysis in HEYA8 cells showing efficiency of YAP1 knockdown on the RNA and protein level using two different siRNAs ( n = 3). h Bar graphs showing number of dead (SYTOX Red positive, black ) and living (SYTOX Red negative, red ) HEYA8 cells after 72 h of low attachment and 96 h after siRNA transfection ( n = 3, two-sided Student’s t -test). i QRT–PCR and western blot analysis in OVCAR8 cells showing efficiency of YAP1 knockdown on the RNA and protein level using two different siRNAs ( n = 3). j Bar graphs showing number of dead (SYTOX Red positive, black ) and living (SYTOX Red negative, red ) OVCAR8 cells after 72 h of low attachment and 96 h after siRNA transfection ( n = 3, two-sided Student’s t -test). Bars and error bars represent mean values and the corresponding SEMs (* p

Article Snippet: Quantative reverse transcription –PCR analysis For mRNA quantification, total RNA was isolated using the Direct-zol RNA MiniPrep kit (Zymo Research Corp.) and cDNAs were synthesized using the Verso cDNA kit (Thermo Scientific) per the manufacturer’s instructions.

Techniques: Western Blot, Incubation, Immunofluorescence, Staining, Quantitative RT-PCR, Transfection

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: 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

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

Journal: Nature Communications

Article Title: Epigenetic memory of the first cell fate decision prevents complete ES cell reprogramming into trophoblast

doi: 10.1038/ncomms6538

Figure Lengend Snippet: The three core TS cell transcription factors Cdx2, Elf5 and Tead4 are insufficient to complete reprogramming to induce a stable TS cell phenotype in bulk culture. ( a ) iCdx2 cells, shown previously to be capable of placental contribution 18 , and iCdx2/iRaf cells were transfected with an Elf5-2A-Tead4-2A-Hygro R (E+T) or Hygro R -only control (vec) expression construct, subjected to the trans-differentiation protocol for 6 days on antibiotic-resistant MEFs, FACS-purified from MEFs and assessed for gatekeeper gene expression levels by RT–qPCR. Details of overexpressed genes resulting from the various conditions are given in red. Elf5 and Tead4 expression, in conjunction with Cdx2 activation, improves the activation of some of the remaining gatekeeper genes. Values are of three biological replicates and expressed as percent of TS cell expression levels combined of the two independent TS cell lines (mean±s.e.m.). ( b ) Activation of TS cell transcription factors in the same cells as in ( a ). Hyperactivation of Tcfap2c and Hand1 (in a ) indicate onset of terminal trophoblast differentiation. Values expressed as in a . ( c ) Dynamics of methylation reprogramming of these cells at the ES-DMRs identified by meDIP-seq and assessed by Sequenom analysis as in Fig. 4c . ( d ) Assessment of phenotypic stability of reprogrammed TSL (iCdx2) cells. Upon removal of the MEF layer, the tight epithelial colony shape that is characteristic of bona fide TS cells was rapidly lost, and cells failed to maintain the co-expression of Cdx2 and Elf5 that is required to preserve the proliferative, self-renewing TS cell state, and instead started to differentiate. Scale bars: 200 μm (phase contrast), 100 μm (immunofluorescence). ( e ) RNA-seq analysis of iCdx2 (5ECER4G20) and iCdx2/iRaf ES cells grown for 5 days on MEFs, and then replated for 3 days on gelatinized TC plastic. Note the instability of gene expression, and specifically the downregulation of critical TS cell genes such as Eomes , Elf5 , Esrrb and Sox2 upon transfer of reprogrammed TSL cells from MEFs onto TC plastic.

Article Snippet: Cells were FACS-purified to separate them from MEFs, and total RNA prepared using TRI reagent (Sigma T9424) followed by DNase treatment using the TURBO DNA-free kit (Life Technologies AM1907), according to the manufacturers’ instructions. mRNA was isolated from total DNA-free RNA (150–240 ng) using the Dynabeads mRNA purification kit (Life Technologies 61006) and prepared into an indexed, strand-specific library using the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre SSV21106) according to the manufacturers’ instructions.

Techniques: Transfection, Expressing, Construct, FACS, Purification, Quantitative RT-PCR, Activation Assay, Methylation, Methylated DNA Immunoprecipitation, Immunofluorescence, RNA Sequencing Assay

Journal: Nature Communications

Article Title: Epigenetic memory of the first cell fate decision prevents complete ES cell reprogramming into trophoblast

doi: 10.1038/ncomms6538

Figure Lengend Snippet: Selection strategy by epithelial morphology in the most efficient iCdx2/iRaf reprogramming model. ( a ) Phase contrast images of TSL clones selected on the basis of colony shape and epithelial morphology. After an initial 5-day reprogramming on MEFs, cells were passaged for 5 weeks on tissue culture plastic in TS cell conditions in the presence of 4HT, during which time colonies were picked and expanded. Clone heterogeneity is observed, with clones CRVT3 and CRVT6 exhibiting the most genuinely TS-like epithelial colony shape. Scale bar: 200 μm. ( b ) Expression analysis of a cohort of trophoblast stem cell and differentiation-associated genes, as well as the identified gatekeeper loci, in selected clones. After growing/expanding colonies in the presence of 4HT for 5 weeks, cells were seeded ±4HT and RNA harvested after 2 days. Tight epithelial morphology in clones CRVT3 and CRVT6 correlates with TS-like Eomes expression levels. Overall, however, differentiation-associated markers are vastly upregulated, and gatekeeper gene expression remains unbalanced. Lack of transcriptional activation of Elf5 , Map3k8 , Sh2d3c and Tead4 is evident. Values are of three replicates and expressed as mean±s.e.m. ( c ) Immunostaining on these clones for Cdx2 and Elf5. Frequent lack of activation or re-silencing of Elf5, correlated with a more pronounced tendency to differentiate, is observed. ( d ) Immunostaining for TS cell surface markers Cd40 and Plet1. Plet1 is a GPI-anchored membrane-associated protein in TS cells, but is redistributed towards a cytoplasmic localization in differentiating trophoblast cells (arrowhead). Despite the TS-like colony shape, reprogrammed cell clones are extremely variable for Cd40 and largely fail to express Plet1 on their cell surface in a TS-like pattern and intensity. All photographs in c , d were taken at precisely the same exposure settings. Scale bars: 100 μm.

Article Snippet: Cells were FACS-purified to separate them from MEFs, and total RNA prepared using TRI reagent (Sigma T9424) followed by DNase treatment using the TURBO DNA-free kit (Life Technologies AM1907), according to the manufacturers’ instructions. mRNA was isolated from total DNA-free RNA (150–240 ng) using the Dynabeads mRNA purification kit (Life Technologies 61006) and prepared into an indexed, strand-specific library using the ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre SSV21106) according to the manufacturers’ instructions.

Techniques: Selection, Clone Assay, Expressing, Activation Assay, Immunostaining

Journal: Molecular plant-microbe interactions : MPMI

Article Title: The Agrobacterium F-Box Protein Effector VirF Destabilizes the Arabidopsis GLABROUS1 Enhancer/Binding Protein-Like Transcription Factor VFP4, a Transcriptional Activator of Defense Response Genes

doi: 10.1094/MPMI-07-17-0188-FI

Figure Lengend Snippet: VFP4-controlled genes ATL31 and At2g32030 . Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of ATL31 and At2g32030 expression in roots of A , wild-type Col-0 and vfp4-1 plants and B , wild-type Col-0 and VFP4 OE-6 plants. C , RT-qPCR analysis of ATL31 and At2g32030 expression in roots of the wild-type Col-0, ATL31 OE-4, and ATL31 OE-5 and Col-0 and At2g32030 OE-9 plants, respectively. The levels of expression were normalized to the internal reference genes ACT7 and 18S RNA . The expression level of each tested gene in the wild-type Col-0 is set to 1.0, and error bars represent standard error of the mean of independent biological replicates, n = 3.

Article Snippet: For RT-PCR analysis of VFP4 expression in vfp4-1 plants, total RNA was extracted from leaves of the wild-type Col-0 and vfp4-1 plants using TRIzol (Invitrogen) and was purified with the SV total RNA isolation system (Promega).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

Journal: Molecular plant-microbe interactions : MPMI

Article Title: The Agrobacterium F-Box Protein Effector VirF Destabilizes the Arabidopsis GLABROUS1 Enhancer/Binding Protein-Like Transcription Factor VFP4, a Transcriptional Activator of Defense Response Genes

doi: 10.1094/MPMI-07-17-0188-FI

Figure Lengend Snippet: VFP4 localizes to the nucleus and cytoplasm. A , Subcellular localization of VFP4. Location of the cell nucleus is indicated by a white arrowhead. All images are projections of single confocal sections and are representative images of three independent experiments. Scale bar = 20 μm. B , Real-time quantitative polymerase chain reaction analysis of VFP4 gene expression following inoculation of wild-type plants by agrobacterium. The levels of expression were normalized to those of ACT7 and 18S RNA . The expression level of VFP4 in the mock-inoculated plants is set to 1.0, and error bars represent standard error of the mean of independent biological replicates, n = 3.

Article Snippet: For RT-PCR analysis of VFP4 expression in vfp4-1 plants, total RNA was extracted from leaves of the wild-type Col-0 and vfp4-1 plants using TRIzol (Invitrogen) and was purified with the SV total RNA isolation system (Promega).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Molecular plant-microbe interactions : MPMI

Article Title: The Agrobacterium F-Box Protein Effector VirF Destabilizes the Arabidopsis GLABROUS1 Enhancer/Binding Protein-Like Transcription Factor VFP4, a Transcriptional Activator of Defense Response Genes

doi: 10.1094/MPMI-07-17-0188-FI

Figure Lengend Snippet: Gain-of-function VFP4 OE-6 and VFP4 OE-14 plants exhibit reduced susceptibility to Agrobacterium tumorigenicity. A , Real-time quantitative polymerase chain reaction analysis of VFP4 gene expression in roots of wild-type Col-0, VFP4 OE-6, and VFP4 OE-14 plants. The levels of expression were normalized to the internal reference genes ACT7 and 18S RNA . The expression level of VFP4 in the wild-type Col-0 was set to 1.0, and error bars represent standard error of the mean (SEM) of independent biological replicates, n = 3. B , Tumors developed on root explants inoculated with A. tumefaciens . C , Quantification of tumorigenicity. Error bars represent SEM of n = 3 biological replicates. Differences in tumorigenicity values between wild-type Col-0 and VFP4 OE-14 plants indicated by different letters are statistically significant ( P values

Article Snippet: For RT-PCR analysis of VFP4 expression in vfp4-1 plants, total RNA was extracted from leaves of the wild-type Col-0 and vfp4-1 plants using TRIzol (Invitrogen) and was purified with the SV total RNA isolation system (Promega).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: bioRxiv

Article Title: BSU1 family phosphatases mediate Flagellin-FLS2 signaling through a specific phosphocode

doi: 10.1101/685610

Figure Lengend Snippet: BSU1 family phosphatases are required for flg22-induced immune responses. a) Identification of flg22-induced BSU1 S251 phosphorylation by SILIA IP-MS using PRM analysis. b) The locus and conservation of BSU1 S251 residue. Asterisk denotes BSU1 S251. c) Flg22-induced MPK3/6 phosphorylation in wild-type (WT) and bsu-q . Eight-week short-day grown leaves were treated with 100 nM flg22 for 2, 5, 10 and 30 min. Fifty µg of protein extracts were loaded to 10% SDS-PAGE gel. d) Flg22-induced MPK3/6 phosphorylation of WT and bsu-i . Nine-day old seedlings grown on 20 uM estradiol or the same amount of DMSO were treated with 100 nM flg22 for 10 min. Immunoblot was performed using anti-phospho-p44/42 MAPK antibody. e) Heat map of log 2 fold change values of flg22-responsive genes in WT and bsu-q from RNA-seq data. f) Scatter plot of log 2 fold change values of flg22-regulated genes in WT vs. bsu-q . Total 1590 genes from flg22-regulated genes in WT (-2≥ FC ≥2) were compared to those in bsu-q . g) qRT-PCR of flg22-induced FRK1 and At2g17740 expression in WT and bsu-q . h) Flg22-induced bacterial growth inhibition in WT and bsu-q . Each replicate includes four leaf discs and 12 biological replicates were used, and the experiment was repeated at least three times with similar results. The average bacterial titer ±SD is reported.

Article Snippet: RNA-seq analysis Total RNA was extracted from the wild-type and bsu-q seedlings treated with flg22 or mock solution using the Spectrum Plant Total RNA kit (Sigma). cDNA libraries were constructed using a TruSeq RNA sample preparation kit (Illumina) according to the manufacturer’s instruction.

Techniques: SDS Page, RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Inhibition

Journal: PLoS ONE

Article Title: High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos

doi: 10.1371/journal.pone.0172171

Figure Lengend Snippet: Bioanalyzer Agilent electrophoresis runs. Examples of representative Bioanalyzer Agilent electrophoresis runs for the five different methods applied for RNA extractions from P . lividus embryos: TRIzol, GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) and Aurum™ Total RNA Mini Kit (Biorad). Four different numerical amounts of embryos were used for RNA extraction: 500, 1000, 2500 and 5000 embryos. The ladder (L) is reported in the first lane of each run. The green band at the bottom of each panel is the RNA 6000 Nano Marker (Agilent RNA 6000 Nano Kit, Agilent Technologies, Inc.). Red box in the 500 lane of TRIzol indicates that the Bioanalyzer software cannot calculate RIN values (reported as N/A in the Table 1 ) for this sample, because of very low concentration and high level of degradation of the RNA.

Article Snippet: We fertilized four pools of eggs and performed RNA extractions from embryos at the pluteus stage (at 48 hour post-fertilization) preserved in RNAlater ® , using five different protocols: a guanidinium-thiocyanate-phenol-chloroform (GTPC) extraction protocol with TRIzol® , and four widely-used Silica Membrane kits, namely GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) andAurum™ Total RNA Mini Kit (Biorad).

Techniques: Electrophoresis, RNA Extraction, Marker, Software, Concentration Assay

Journal: PLoS ONE

Article Title: High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos

doi: 10.1371/journal.pone.0172171

Figure Lengend Snippet: Agilent Bioanlyzer electropherograms. Examples of representative Agilent Bioanlyzer electropherograms of P . lividus RNA: for TRIzol, GenElute and RNAqueous RNA extraction from 5000 embryos extraction; for RNeasy and Aurum RNA extraction from 2500 embryos (see also Table 1 ). Relative Fluorescent Unit (FU) and seconds of migration (s) of RNA samples isolated according to the five different extraction methods are reported. RIN values are also reported.

Article Snippet: We fertilized four pools of eggs and performed RNA extractions from embryos at the pluteus stage (at 48 hour post-fertilization) preserved in RNAlater ® , using five different protocols: a guanidinium-thiocyanate-phenol-chloroform (GTPC) extraction protocol with TRIzol® , and four widely-used Silica Membrane kits, namely GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) andAurum™ Total RNA Mini Kit (Biorad).

Techniques: RNA Extraction, Migration, Isolation

Journal: BMC Genomics

Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

doi: 10.1186/s12864-019-5608-2

Figure Lengend Snippet: Sorting small cell populations directly into the lysis buffer of the RNA isolation kit improves RNA quality and yield. ( a ) RQN values ( b ) and RNA yield of RNA samples isolated from a range of cell numbers (5000–200,000) when sorting directly into the lysis buffer of the RNA isolation kit or collecting the cells first into a collection medium. RQN values ( c ) and RNA yield ( d ) of RNA samples isolated from a range of cell numbers (5000–200,000) sorted directly into the lysis buffer or sorted into a collection buffer first. But here the volume of the lysis buffer was amended not to exceed its maximum dilution point caused by the sorting procedure. Left panels show samples isolated with the RNeasy plus micro kit, right panels with the RNAqueous micro kit. Average of two biological replicates is shown. Error bars indicate SEM

Article Snippet: RNA isolation and quality control RNA from sorted cells was isolated with the use of the RNeasy plus micro kit (Qiagen, 74,034) or the RNAqueous-Micro Total RNA Isolation Kit (Ambion, AM1931).

Techniques: Lysis, Isolation

Journal: BMC Genomics

Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

doi: 10.1186/s12864-019-5608-2

Figure Lengend Snippet: Sorting cells directly into the lysis buffer preserves and protects RNA from degradation. RQN values of individual RNA samples isolated at specific time points after FACS sorting of 20,000 fli1a:EGFP cells. Cells were sorted directly in the lysis buffer of the kit or first captured into a collection medium. Left panel shows samples isolated with the RNAqueous micro kit, right panel with the RNeasy plus micro kit

Article Snippet: RNA isolation and quality control RNA from sorted cells was isolated with the use of the RNeasy plus micro kit (Qiagen, 74,034) or the RNAqueous-Micro Total RNA Isolation Kit (Ambion, AM1931).

Techniques: Lysis, Isolation, FACS

Journal: BMC Genomics

Article Title: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

doi: 10.1186/s12864-019-5608-2

Figure Lengend Snippet: Comparative qualitative analysis of RNA purified with the Rnaqeuous and Rneasy micro kit. ( a ) Boxplots showing RQN values and ( b ) RNA yield of 10 RNA samples purified from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent RQN values of individual samples. ( c ) Boxplots showing 5′-3′ delta-Cq (dCq) values calculated from a 5′ and a 3′ RT-qPCR assay of 7 RNA samples isolated from 20,000 FACS sorted fli1a:EGFP zebrafish cells with the RNAqueous micro ( left ) and the RNeasy plus micro ( right ) kit. Red dots represent 5′-3′ dCq values of individual samples. * P

Article Snippet: RNA isolation and quality control RNA from sorted cells was isolated with the use of the RNeasy plus micro kit (Qiagen, 74,034) or the RNAqueous-Micro Total RNA Isolation Kit (Ambion, AM1931).

Techniques: Purification, FACS, Quantitative RT-PCR, Isolation

Journal: eLife

Article Title: FMRP has a cell-type-specific role in CA1 pyramidal neurons to regulate autism-related transcripts and circadian memory

doi: 10.7554/eLife.46919

Figure Lengend Snippet: Optimization of TRAP immunoprecipitation. The amount of antibody required for the TRAP IP was determined by titration. 500 μl hippocampal lysate was incubated with varying amounts of anti-HA antibody from 2.5 to 12.5 μg, followed by pull down of the antibody-ribosome-mRNA complex with protein A dynabeads. After washing, half the beads were used for protein extraction while the other half were used RNA isolation. ( A ) Protein concentration in the input lysate and remaining in the supernatant after IP (Post-IP Lysate) as well as the amount of protein eluted from the beads was determined by Western blotting. Western blots for the ribosomal protein RPP0 and the HA-tagged RPL22 ribosomal protein are shown (i) along with the quantification of the protein bands (ii). ( B ) Concentration of RNA eluted from the beads is shown for each IP. Based on these combined results 10 μg antibody per 500 μl lysate was determined to be the optimal concentration to ensure maximal depletion of the HA-tagged polysomes and give the best signal-to-noise.

Article Snippet: Sequencing libraries were prepared using Poly(A) RNA selection (Dynabeads mRNA Purification Kit, ThermoFisher Scientific) and the TruSeq RNA Library Prep Kit (Illumina).

Techniques: Immunoprecipitation, Titration, Incubation, Protein Extraction, Isolation, Protein Concentration, Western Blot, Concentration Assay

Journal: PLoS ONE

Article Title: High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos

doi: 10.1371/journal.pone.0172171

Figure Lengend Snippet: Bioanalyzer Agilent electrophoresis runs. Examples of representative Bioanalyzer Agilent electrophoresis runs for the five different methods applied for RNA extractions from P . lividus embryos: TRIzol, GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) and Aurum™ Total RNA Mini Kit (Biorad). Four different numerical amounts of embryos were used for RNA extraction: 500, 1000, 2500 and 5000 embryos. The ladder (L) is reported in the first lane of each run. The green band at the bottom of each panel is the RNA 6000 Nano Marker (Agilent RNA 6000 Nano Kit, Agilent Technologies, Inc.). Red box in the 500 lane of TRIzol indicates that the Bioanalyzer software cannot calculate RIN values (reported as N/A in the Table 1 ) for this sample, because of very low concentration and high level of degradation of the RNA.

Article Snippet: We fertilized four pools of eggs and performed RNA extractions from embryos at the pluteus stage (at 48 hour post-fertilization) preserved in RNAlater ® , using five different protocols: a guanidinium-thiocyanate-phenol-chloroform (GTPC) extraction protocol with TRIzol® , and four widely-used Silica Membrane kits, namely GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) andAurum™ Total RNA Mini Kit (Biorad).

Techniques: Electrophoresis, RNA Extraction, Marker, Software, Concentration Assay

Journal: PLoS ONE

Article Title: High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos

doi: 10.1371/journal.pone.0172171

Figure Lengend Snippet: Agilent Bioanlyzer electropherograms. Examples of representative Agilent Bioanlyzer electropherograms of P . lividus RNA: for TRIzol, GenElute and RNAqueous RNA extraction from 5000 embryos extraction; for RNeasy and Aurum RNA extraction from 2500 embryos (see also Table 1 ). Relative Fluorescent Unit (FU) and seconds of migration (s) of RNA samples isolated according to the five different extraction methods are reported. RIN values are also reported.

Article Snippet: We fertilized four pools of eggs and performed RNA extractions from embryos at the pluteus stage (at 48 hour post-fertilization) preserved in RNAlater ® , using five different protocols: a guanidinium-thiocyanate-phenol-chloroform (GTPC) extraction protocol with TRIzol® , and four widely-used Silica Membrane kits, namely GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) andAurum™ Total RNA Mini Kit (Biorad).

Techniques: RNA Extraction, Migration, Isolation

Journal: PLoS ONE

Article Title: GPR158, an Orphan Member of G Protein-Coupled Receptor Family C: Glucocorticoid-Stimulated Expression and Novel Nuclear Role

doi: 10.1371/journal.pone.0057843

Figure Lengend Snippet: GPR158 regulates the proliferation of TBM-1 cells. TBM-1 cells were transfected at 70–80% confluence using Lipofectamine LTX reagent with either GPR158 expression plasmids or vector alone ( A ) OR either GPR158 siRNA or control scrambled siRNA as indicated ( B and C ) and incubated in growth medium for 3 days in a 6-well culture dishes. ( A and C ) After 3 days of transfection, the cells were trypsinized and counted using trypan blue dye in a hemocytometer chamber for the cells transfected with indicated plasmids. ( B ) Total RNA was isolated for analyzing the levels of GPR158 mRNA by qRT-PCR. β-actin was used as a reference gene. ( A, B and C ) The data represent mean ± SEM of three independent experiments. ***P

Article Snippet: Quantitative real-time reverse transcription-PCR (qRT-PCR) Total RNA was isolated using Aurum total RNA mini kit (Bio-Rad, Hercules, CA) and qRT-PCR analysis was carried out using iScript one-step RT-PCR kit with SYBR Green (Bio-Rad) on an ABI PRISM 7900 HT sequence detection system (Applied Biosystems, Foster City, CA), according to the manufacturer's instructions.

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Isolation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: GPR158, an Orphan Member of G Protein-Coupled Receptor Family C: Glucocorticoid-Stimulated Expression and Novel Nuclear Role

doi: 10.1371/journal.pone.0057843

Figure Lengend Snippet: Expression and GC-mediated induction of GPR158 in trabecular meshwork cells. ( A ) RT-PCR analysis of GPR158, aquaporin-1, myocilin and β-actin mRNA expression in TBM-1 cells. ( B ) Western blotting for GPR158 in cellular extracts from untreated and Dex treated (250 nM) for 6 days using anti-C-terminal GPR158 antibodies (1∶1000 dilution). The data are representative of three independent experiments. The vertical line indicates repositioned gel lanes. ( C and D ) TBM-1 cells were stimulated with either Dex ( C ) or TA ( D ) for the indicated time periods. Total RNA was isolated for quantitation of GPR158 mRNA by qRT-PCR. GAPDH was used as a reference gene. The cells treated with ethanol (0.1%) were used as a negative control. The data are representative of three independent experiments. ***P

Article Snippet: Quantitative real-time reverse transcription-PCR (qRT-PCR) Total RNA was isolated using Aurum total RNA mini kit (Bio-Rad, Hercules, CA) and qRT-PCR analysis was carried out using iScript one-step RT-PCR kit with SYBR Green (Bio-Rad) on an ABI PRISM 7900 HT sequence detection system (Applied Biosystems, Foster City, CA), according to the manufacturer's instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Isolation, Quantitation Assay, Quantitative RT-PCR, Negative Control

Journal: Scientific Reports

Article Title: Probing the phenomenon of trained immunity in invertebrates during a transgenerational study, using brine shrimp Artemia as a model system

doi: 10.1038/srep21166

Figure Lengend Snippet: Vibrio campbellii -exposed parental generation and their unexposed progenies showed increased expression of hsp70 and hmgb1 genes. See Fig. 9 for explanation of the experimental groups. Total RNA extracted from F0 to F3 juveniles, reared under common garden conditions, was analyzed for expression of ( A ) hsp70 and ( B ) hmgb1 genes by qPCR assay. At each generation, the expression of hsp70 or hmgb1 gene in the control group was set at 1.0 and all other data points were normalized accordingly using the equation of Pfaffl et al. (2002) 52 . The actin gene was used as an internal control. Error bars represent the standard errors of three biological replicates. Significant differences between the treatment and control at respective generation are indicated by *(P

Article Snippet: RNA extraction and quantitative real-time PCR (RT-qPCR) analysis Total RNA was extracted from the Artemia samples using the SV total RNA isolation kit (Promega, Belgium).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: BMC Genomics

Article Title: An optimized protocol for generation and analysis of Ion Proton sequencing reads for RNA-Seq

doi: 10.1186/s12864-016-2745-8

Figure Lengend Snippet: Ion Proton RNA-Seq library preparation workflow. a Life Technologies’ protocol: mRNAs were fragmented by RNaseIII using the Ion Total RNA-Seq Kit V2, only the sense RNA strands (mRNA) were sequenced. b BGI’s protocol: based on chemical (heat) fragmentation. Additional size selection steps were introduced after adaptor ligation

Article Snippet: Library preparation and sequencing statistics Two libraries were constructed by RNaseIII fragmentation according to Ion Total RNA-Seq Kit v2 specifications (Life Technologies, Fig. ), the other nine were by chemical fragmentation according to the BGI protocol (Fig. ), yielding a total of more than 204 million reads.

Techniques: RNA Sequencing Assay, Selection, Ligation

Journal: Human genetics

Article Title: A zebrafish model of foxe3 deficiency demonstrates lens and eye defects with dysregulation of key genes involved in cataract formation in humans

doi: 10.1007/s00439-018-1884-1

Figure Lengend Snippet: Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) shows upregulation of dnajb1b in foxe3 indel mutant larvae RNA was extracted at 3 days post fertilization (dpf) from pools of control larvae and larvae with lens defects (putative homozygotes) obtained from an incross of fish that were heterozygous for an indel variant in foxe3 . After cDNA synthesis, two pairs of gene-specific primers were used to amplify dnajb1a and dnjab1b and one pair of gene-specific primers was used to amplify foxe3 . qRT-PCR was performed and analyzed using the ΔΔCt method and gapdh as an internal control gene. The results show log2 expression fold change plotted for dnajb1a , dnajb1b , and foxe3 and, in addition to confirming downregulation of foxe3 , demonstrate upregulation of dnajb1b ( p = 0.076), but not dnajb1a , in larvae with eye defects from the foxe3 indel mutant incross. All values are an average of 3 biological replicates with 3 replicates for each experiment and the error bars indicate the standard error of the mean.

Article Snippet: After designing gene-specific primers for dnajb1a and dnajb1b , we obtained total RNA (Qiagen RNeasy kit) at 3 dpf from wildtype EKW larvae and larvae with eye defects selected from a heterozygous, foxe3 indel incross as described above.

Techniques: Polymerase Chain Reaction, Quantitative RT-PCR, Mutagenesis, Fluorescence In Situ Hybridization, Variant Assay, Expressing

Journal: Human genetics

Article Title: A zebrafish model of foxe3 deficiency demonstrates lens and eye defects with dysregulation of key genes involved in cataract formation in humans

doi: 10.1007/s00439-018-1884-1

Figure Lengend Snippet: RNA-Seq analysis of foxe3 indel mutant zebrafish A: Principal component analysis (PCA) plot analysis of foxe3 indel mutant and wildtype RNA-Seq datasets. Principal component analysis (PCA) of foxe3 indel mutant and wildtype RNA-Seq datasets showed mutant (1, 2 and 3) and wildtype (4, 5 and 6) datasets as distinct clusters. B: Differentially expressed genes in foxe3 indel mutant compared to control. A plot of fold change (FC) for differentially expressed genes in foxe3 indel mutant against mean expression levels in fragments per kilobase of transcript per million mapped reads (FPKM), highlighting downregulated (green) and upregulated (red) genes at 3 dpf.

Article Snippet: After designing gene-specific primers for dnajb1a and dnajb1b , we obtained total RNA (Qiagen RNeasy kit) at 3 dpf from wildtype EKW larvae and larvae with eye defects selected from a heterozygous, foxe3 indel incross as described above.

Techniques: RNA Sequencing Assay, Mutagenesis, Expressing

Journal: Journal of microbiological methods

Article Title: Comparative Evaluation of cDNA Library Construction Approaches for RNA-Seq Analysis from Low RNA-Content Human Specimens

doi: 10.1016/j.mimet.2018.10.008

Figure Lengend Snippet: Overlap of the S. sanguinis expressed non-rRNA genes between the two library generation methods using the sonicate fluid sample (a) and the synovial fluid sample (b); and between the two sample types processed by the Illumina TruSeq Stranded Total RNA kit (c) and the NuGEN Ovation SoLo RNA-Seq System (d).

Article Snippet: In this study, two strand-specific cDNA library construction kits, the Ovation SoLo RNA-Seq System (NuGEN Technologies) and the TruSeq Stranded Total RNA (Illumina), were compared for their ability to generate informative cDNA libraries for RNA-Seq from samples from a patient with periprosthetic joint infection, an infection-type with a low microbial burden.

Techniques: RNA Sequencing Assay

Journal: International Journal of Molecular Sciences

Article Title: Transcriptome Analysis Reveals Molecular Signatures of Luteoloside Accumulation in Senescing Leaves of Lonicera macranthoides

doi: 10.3390/ijms19041012

Figure Lengend Snippet: Transcriptomic mapping of genes associated with luteoloside biosynthesis in Lonicera macranthoides . Proposed pathways for luteoloside biosynthesis in Lonicera macranthoides were illustrated by RNA-Seq analysis. Luteolin, the precursor of luteoloside, is biosynthesized from the general flavonoid precursor: naringenin. Circle 1(①) indicates that luteolin is biosynthesized directly from apigenin catalyzed by F3’H. Circle 2(②) indicates that luteolin is generated directly from eriodictyol catalyzed by FNS. Expression profile for each gene was shown in colored blocks and each blocks represented the expression changes (represented by Log2Ratio) in senescing leaves with respect to young leaves. Red colors/green colors correspond to up-/down-regulation of these genes and Log2Ratio ≥ 1 is considered statistically significant. Details were showed in Table 2 . Abbreviations: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxycinnamoyl CoA ligase/4-coumarate-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; FNS, flavone synthase; F3H, flavonoid 3′-monooxygenase/flavonoid 3′-hydroxylase; UF7GT, flavone 7- O -β-glucosyltransferase.

Article Snippet: RNA Extraction and Illumina Sequencing Total RNA from young and senescing leaves was isolated and purified using QIAGEN RNeasy Plant Mini kit and RNase-free DNase set (QIAGEN, Hilden, Germany) according to manufacturer’s instruction.

Techniques: RNA Sequencing Assay, Generated, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Transcriptome Analysis Reveals Molecular Signatures of Luteoloside Accumulation in Senescing Leaves of Lonicera macranthoides

doi: 10.3390/ijms19041012

Figure Lengend Snippet: Expression patterns of selected unigenes related to luteoloside biosynthesis identified by RNA-Seq were validated by qRT-PCR. Expressions of unigenes located upstream of luteoloside metabolic pathway ( A ) Unigene109136 ( PAL ), ( B ) CL11118.Contig2 ( C4H ), ( C ) Unigene29692 ( 4CL ) and downstream of luteoloside metabolic pathway ( D ) CL11269.Contig3 ( CHI ), ( E ) CL19869.Contig1 ( CHS ), ( F ) Unigene2335 ( FNSII ), ( G ) CL11828.Contig1 and ( H ) CL11828.Contig2 ( F3’H ), ( I ) Unigene2918 and ( J ) Unigene97915 ( UFGT ) in young and senescing leaves were analyzed by qRT-PCR. Relative expression levels were determined based on the reference young leaves set to 1. Values are means ± SD of three biological repetitions ( n = 3). Two technical replicates for each biological replicate were performed in each qRT-PCR experiments.

Article Snippet: RNA Extraction and Illumina Sequencing Total RNA from young and senescing leaves was isolated and purified using QIAGEN RNeasy Plant Mini kit and RNase-free DNase set (QIAGEN, Hilden, Germany) according to manufacturer’s instruction.

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Transcriptome Analysis Reveals Molecular Signatures of Luteoloside Accumulation in Senescing Leaves of Lonicera macranthoides

doi: 10.3390/ijms19041012

Figure Lengend Snippet: Number of differentially expressed unigenes during leaf senescence identified by RNA-Seq data in Lonicera macranthoides . Differentially expressed unigenes (DEGs) between young and senescing leaves were illustrated by bar chart. Red and green bars represent the up-regulated and down-regulated unigenes in senescing leaves (SL) compared with those in young leaves (YL), respectively in Lonicera macranthoides .

Article Snippet: RNA Extraction and Illumina Sequencing Total RNA from young and senescing leaves was isolated and purified using QIAGEN RNeasy Plant Mini kit and RNase-free DNase set (QIAGEN, Hilden, Germany) according to manufacturer’s instruction.

Techniques: RNA Sequencing Assay

Journal: International Journal of Molecular Sciences

Article Title: Transcriptome Analysis Reveals Molecular Signatures of Luteoloside Accumulation in Senescing Leaves of Lonicera macranthoides

doi: 10.3390/ijms19041012

Figure Lengend Snippet: Expression patterns of selected unigenes related to transcription factors identified by RNA-Seq were validated by qRT-PCR. Expressions of unigenes, homologs to AtMYB12 ( A ) Unigene36582 and ( B ) Unigene52460 , homologs to VvMYB75 ( C ) CL5870.Contig1 , homologs to VvMYB1R1 ( D ) CL15118.Contig2 , homologs to AtbHLH113 ( E ) Unigene33227 and ( F ) Unigene11884 , homologs to AtbHLH78 ( G ) CL2495.Contig7 , homologs to TTG1 ( H ) Unigene108470 and homologs to AtMYB5 ( I ) CL20138.Contig2 in young and senescing leaves were analyzed by qRT-PCR. Relative expression levels were determined based on the reference young leaves set to 1. Values are means ± SD of three biological repetitions ( n = 3). Two technical replicates for each biological replicate were performed in each qRT-PCR experiments.

Article Snippet: RNA Extraction and Illumina Sequencing Total RNA from young and senescing leaves was isolated and purified using QIAGEN RNeasy Plant Mini kit and RNase-free DNase set (QIAGEN, Hilden, Germany) according to manufacturer’s instruction.

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR

Journal: Journal of microbiological methods

Article Title: Comparative Evaluation of cDNA Library Construction Approaches for RNA-Seq Analysis from Low RNA-Content Human Specimens

doi: 10.1016/j.mimet.2018.10.008

Figure Lengend Snippet: Overlap of the S. sanguinis expressed non-rRNA genes between the two library generation methods using the sonicate fluid sample (a) and the synovial fluid sample (b); and between the two sample types processed by the Illumina TruSeq Stranded Total RNA kit (c) and the NuGEN Ovation SoLo RNA-Seq System (d).

Article Snippet: For instance, although the Illumina TruSeq Stranded Total RNA kit generated a higher number of microbial reads than the NuGEN Ovation SoLo RNA-Seq System kit, the percentage of these reads indicated less microbial RNA in the Illumina libraries.

Techniques: RNA Sequencing Assay

Journal: PLoS Pathogens

Article Title: An essential EBV latent antigen 3C binds Bcl6 for targeted degradation and cell proliferation

doi: 10.1371/journal.ppat.1006500

Figure Lengend Snippet: EBNA3C regulates Bcl6 mRNA expression through inhibition of its promoter activity. A) 5 million BJAB, BJAB7, BJAB10, LCL1 and LCL2 cells were harvested and extracted total RNA using Trizol reagent. Then cDNA was prepared with reverse transcriptase kit, and detected Bcl6 mRNA expression by quantitative Real-time PCR analysis (SYBR green). GAPDH was set as an internal reference. Each sample was determined in triplicate. B) EBNA3C knock-down (sh-E3C) stable LCL1 or control (sh-Ctrl) LCL1 cells were harvested and Bcl6 mRNA expression was detected using Real-time PCR as mentioned. C) 10 million BJAB10 cells were transfected with specific EBNA3C (sh-E3C) or control (sh-Ctrl) short hairpin RNA. At 48 hours post-transfection, total RNA was extracted, reverse-transcribed, followed by quantitative Real-time PCR analysis. Meanwhile, EBNA3C expression was also detected by western blot analysis. D) HEK293T cells were transfected with the reporter constructs containing wild-type Bcl6 promoter (pLA/B9) and increasing amount of Myc-EBNA3C. Cells were collected and lysed in lysis buffer at 48 hours post-transfection. Luciferase activity was measured according to the dual-luciferase reporter assay kit. Mean values and standard deviations of two independent experiments were presented. Cell lysate was resolved by 10% SDS-PAGE in order to check EBNA3C expression. GAPDH western blot was done as an internal loading control. E) HEK293T cells were transfected with wild-type Bcl6 promoter reporter plasmids in combination with different expression constructs as indicated. Cells were collected and lysed, then the lysate were used to detect luciferase activity as previously described.

Article Snippet: Then total RNA extraction was performed using Trizol reagent (Invitrogen, Inc., Carlsbad, CA) and treated with Dnase I (Invitrogen, Inc., Carlsbad, CA), then cDNA was prepared with Superscript II reverse transcriptase kit (Invitrogen, Inc., Carlsbad, CA) according to the manufacturer’s protocol.

Techniques: Expressing, Inhibition, Activity Assay, Real-time Polymerase Chain Reaction, SYBR Green Assay, Transfection, shRNA, Western Blot, Construct, Proximity Ligation Assay, Lysis, Luciferase, Reporter Assay, SDS Page

Journal: Journal of Experimental Botany

Article Title: A 9 bp cis-element in the promoters of class I small heat shock protein genes on chromosome 3 in rice mediates L-azetidine-2-carboxylic acid and heat shock responses

doi: 10.1093/jxb/erq230

Figure Lengend Snippet: Effect of a protein synthesis inhibitor on accumulation of Oshsp17.3 transcript induced by AZC and HS. Before 5 mM AZC or HS (41°C) treatment, 3-d-old rice seedlings were treated or not with cycloheximide (CHX) (2 μg ml −1 ) for 30 min at 28 C. A 16 ng aliquot of DNase I-treated total RNA was used for RT-PCR. The RT-PCR products of Oshsp17.3 are shown by ethidium bromide staining, and the RT-PCR product of the 18S rRNA was used as an internal PCR control.

Article Snippet: A 16 ng aliquot of DNase I-treated total RNA was used for RT-PCR analyses with use of the Superscript one-step RT-PCR kit (Invitrogen) according to the manufacturer's protocol.

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