Journal: Reproduction (Cambridge, England)

Article Title: SKAP, an outer kinetochore protein, is required for mouse germ cell development

doi: 10.1530/REP-15-0451

Figure Lengend Snippet: SKAP germline-specific expression. (A) Northern blot (upper panel) and RT-PCR (lower panels) analysis of RNA from various tissues of adult mice. (B) RT-PCR (upper panels) and western blot (lower panels) analysis of SKAP expression in juvenile (day 4–30ppt) and adult mouse testes. Insert in the lower panel: day 21 and 30ppt lanes after shorter exposure. F-actin and anti-α tubulin were used as controls for RT-PCR and western blotting respectively. (C) cDNA sequence of Knstrn , the gene encoding SKAP (NM_026412.3). The alternative in-frame start codon (in blue and underlined) in the second exon encodes a protein that is 9 kDa shorter than full-length SKAP. Different colors represent different exons. (D) Top: cartoon representing the two SKAP isoforms. The region upstream of the alternative start codon is in light blue. The black bar indicates the peptide used to produce the anti-SKAP-N-ter antibody. Bottom: western blotting of juvenile (day 14ppt) and adult testis extracts with the anti-SKAP-N-ter antibody (left panel) and the anti-SKAP antibody. (E) Localization of the long (anti-SKAP-N-ter antibody) and short (anti-SKAP antibody) SKAP isoforms in adult testes. Left panels: seminiferous tubules (20× magnification). Right panels: cells at the periphery of a seminiferous tubule (100× magnification). Green arrows, SKAP-positive spermatids; red arrow, SKAP-positive spermatogonia. Scale bar: 10 μm.

Article Snippet: Northern blotting and RT-PCR Total RNA from various mouse tissues was prepared with the Gene Elute Mammalian Total RNA purification kit (Sigma), according to the manufacturer's instructions.

Techniques: Expressing, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Western Blot, Sequencing

Journal: Frontiers in Microbiology

Article Title: The Effects of Nutrient Imbalances and Temperature on the Biomass Stoichiometry of Freshwater Bacteria

doi: 10.3389/fmicb.2017.01692

Figure Lengend Snippet: Changes in the RNA and DNA content (fg cell −1 ) of each strain across temperatures, with different symbols for each level of supply C:P. Error bars represent one standard error of the mean.

Article Snippet: Bacterial samples on the filters, negative control samples (containing all reagents but no bacteria), and standards of RNA (E. coli , Invitrogen) and DNA (calf thymus, Sigma) were prepared for analysis as described in Makino et al. ( ).

Techniques:

Journal: BMC Plant Biology

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

doi: 10.1186/s12870-015-0580-8

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

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

Techniques: Methylation, Polymerase Chain Reaction, Amplification

Journal: BMC Plant Biology

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

doi: 10.1186/s12870-015-0580-8

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

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

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

Journal: Scientific Reports

Article Title: A-to-I RNA editing enzyme ADAR2 regulates light-induced circadian phase-shift

doi: 10.1038/s41598-018-33114-6

Figure Lengend Snippet: ADAR2-mediated A-to-I RNA editing in mouse SCN. ( a ) Direct sequencing chromatograms from RT-PCR products in control (top) and Adar2 -KO (bottom) mouse SCN at CT22. The solid circles indicate the editing sites in the transcripts. ( b ) A-to-I RNA editing level at each editing sites in direct sequencing analysis (mean ± s.e.m.; n = 3).

Article Snippet: Quantitative RT-PCR (qRT-PCR) For quantification of gene expression, total RNA was reverse transcribed by Go Script Reverse Transcriptase (Promega) with both anchored (dT)15 primers and random oligo primers.

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: Scientific Reports

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

doi: 10.1038/srep34850

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

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

Techniques:

Journal: Scientific Reports

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

doi: 10.1038/srep34850

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

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

Techniques: Software, Concentration Assay

Journal: Scientific Reports

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

doi: 10.1038/srep34850

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

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

Techniques:

Journal: Scientific Reports

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

doi: 10.1038/srep34850

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

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

Techniques: Sequencing, Isolation

Journal: RNA

Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

doi: 10.1261/rna.2610405

Figure Lengend Snippet: Overview of the miRNA microarray procedure. RNA species smaller than ~40 nt are purified by a rapid electrophoretic gel fractionation method. miRNAs are 3′-end labeled with poly(A) polymerase, amine-modified nucleotides, and amine-reactive dyes.

Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

Techniques: Microarray, Purification, Fractionation, Labeling, Modification

Journal: RNA

Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

doi: 10.1261/rna.2610405

Figure Lengend Snippet: Comparison between miRNA microarray data and other detection methods. Total RNA was isolated from human bladder, lung, or uterus. miRNA populations in 10 μg of each sample were independently fractionated, labeled, and compared by pairs on two

Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

Techniques: Microarray, Isolation, Labeling

Journal: RNA

Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

doi: 10.1261/rna.2610405

Figure Lengend Snippet: Comparison between enriched and gel purified miRNA fractions. One, 5, or 10 μg of total RNA from human bladder or lung were used to gel purify miRNAs (GP1, GP5, GP10) or to prepare fractions enriched in small RNA (E1, E5, E10). RNA species in

Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

Techniques: Purification

Journal: RNA

Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

doi: 10.1261/rna.2610405

Figure Lengend Snippet: miRNA microarray reproducibility. Total RNA was isolated from a single human prostate sample and a single human colon sample. Each total RNA sample was split into six independent samples (10 μg each). miRNAs in each sample were independently fractionated

Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

Techniques: Microarray, Isolation

Journal: RNA

Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

doi: 10.1261/rna.2610405

Figure Lengend Snippet: miRNA microarray accuracy and precision. ( A ) Self versus self analysis. A human thymus total RNA sample (20 μg) was split in half and miRNAs were independently gel purified, labeled either with Cy3 or Cy5 dyes, and hybridized to the same array

Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

Techniques: Microarray, Purification, Labeling

Journal: RNA

Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

doi: 10.1261/rna.2610405

Figure Lengend Snippet: Validation of miRNA expression data. Total RNA was isolated from the 10 indicated tissues, independent from the ones used in the microarray profiling study (Fig. 6). Expression levels of let-7C and miR- 200B were analyzed by Northern blot (2

Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

Techniques: Expressing, Isolation, Microarray, Northern Blot

Journal: RNA

Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

doi: 10.1261/rna.2610405

Figure Lengend Snippet: miRNA expression profiles across 26 human normal tissues. miRNAs from 10 μg of the indicated total RNA samples were gel purified, labeled with Cy5, and compared on 26 independent microarrays against Cy3-labeled miRNAs isolated from 10 μg

Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

Techniques: Expressing, Purification, Labeling, Isolation

Journal: PLoS ONE

Article Title: Chronic Oxidative Stress Increases Growth and Tumorigenic Potential of MCF-7 Breast Cancer Cells

doi: 10.1371/journal.pone.0087371

Figure Lengend Snippet: Real time quantitative reverse transcription PCR analysis of gene expression of cell growth and survival related genes. Total RNA isolated from MCF-7 cells with acute and chronic exposure to H 2 O 2 was used to perform one step real-time quantitative reverse transcription PCR as described in materials and methods. Cycle threshold value (Ct value) of each gene was normalized to the Ct value of housekeeping gene GAPDH obtained from the same sample. The gene expression in fold change was calculated and histogram was plotted using the means of triplicate values. Results of acute (Figure 4A) and chronic (Figure 4B) exposure were presented in separate histograms. Statistically significant change (p

Article Snippet: Single step qRT-PCR amplifications were performed in an iCycler IQ (Bio-Rad) real time PCR machine using one-step RT-PCR kit with SYBR green and 200 ng total RNA following the manufacturer's protocol (Bio-Rad).

Techniques: Polymerase Chain Reaction, Expressing, Isolation

Journal: PLoS ONE

Article Title: Chronic Oxidative Stress Increases Growth and Tumorigenic Potential of MCF-7 Breast Cancer Cells

doi: 10.1371/journal.pone.0087371

Figure Lengend Snippet: Real time quantitative reverse transcription PCR analysis of gene expression of metastasis related genes. Total RNA isolated from MCF-7 cells with acute and chronic exposure to H 2 O 2 was used to perform one step real-time quantitative reverse transcription PCR as described in materials and methods. Cycle threshold value (Ct value) of each gene was normalized to the Ct value of housekeeping gene GAPDH obtained from the same sample. The gene expression in fold change was calculated and histogram was plotted using the means of triplicate values. Results of acute (Figure 5A) and chronic (Figure 5B) exposure were presented in separate histograms. Statistically significant change (p

Article Snippet: Single step qRT-PCR amplifications were performed in an iCycler IQ (Bio-Rad) real time PCR machine using one-step RT-PCR kit with SYBR green and 200 ng total RNA following the manufacturer's protocol (Bio-Rad).

Techniques: Polymerase Chain Reaction, Expressing, Isolation

Journal: Nature

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

doi: 10.1038/s41586-018-0084-4

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

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

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

Journal: PLoS ONE

Article Title: MicroRNA-137 Upregulation Increases Bladder Cancer Cell Proliferation and Invasion by Targeting PAQR3

doi: 10.1371/journal.pone.0109734

Figure Lengend Snippet: The expression of PAQR3 partially rescued miR-137-enhanced cell proliferation and invasion. (A) Western blot analysis showed that transfection of small interfering RNA against PAQR3 into T24 cells led to dramatically decreased PAQR3 protein expression. GAPDH was also detected as a loading control. (B) Silencing of PAQR3 significantly enhanced the proliferation of bladder cancer cells. The growth index as assessed at 0, 24, 48 and 72 h. (C) Western blot analysis of PAQR3 in T24 cells co-transfected with either miR-137 mimic or scramble and 2.0 µg pCDNA- PAQR3 or pCDNA empty vector. GAPDH was also detected as a loading control. (D) Cell growth curves in T24 cells transfected with different combinations at 0, 24, 48 and 72 h. (E) Transwell analysis of T24 cells treated with different combinations. The relative ratio of invasive cells per field is shown right. *p

Article Snippet: For the reporter gene assay, the cells were cotransfected with 0.5 µg of pGL3-PAQR3-3′UTR or pGL3- PAQR3-3′UTR Mut plasmid, 0.05 ng of the phRL-SV40 control vector (Promega, USA), and 100 nM miR-137 or control RNA using Lipofectamine 2000 (Invitrogen, USA).

Techniques: Expressing, Western Blot, Transfection, Small Interfering RNA, Plasmid Preparation

Journal: BMC Veterinary Research

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

doi: 10.1186/s12917-017-1218-x

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

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

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

Journal: BMC Veterinary Research

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

doi: 10.1186/s12917-017-1218-x

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

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

Techniques: Incubation, Quantitative RT-PCR

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0162253

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

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

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

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

Article Title: The kinesin KIF17b and RNA-binding protein TB-RBP transport specific cAMP-responsive element modulator-regulated mRNAs in male germ cells

doi: 10.1073/pnas.2536695100

Figure Lengend Snippet: ( A ) Coimmunoprecipitation of KIF17b and TB-RBP from mouse testis extracts. A silver-stained SDS/polyacrylamide gel shows total extracts from brain (lane 1) and testis (lane 2), protein A-agarose beads preclearing controls of extracts from brain (lane 3) and testis (lane 4), and immunoprecipitated cytoplasmic S100 extracts from brain (lane 5) and testis (lane 6) using affinity-purified anti-TB-RBP. Excised protein bands were sequenced by MALDI-TOF mass spectrometry. M, marker proteins. ( B ) KIF17b expression is specific to postmeiotic male germ cells. Shown is a Northern blot of RNA (10 μg) from brain (lane 1), testis (lane 2), mixed germ cells (lane 3), pachytene spermatocytes (lane 4), round spermatids (lane 5), and elongating spermatids (lane 6). Robust expression of KIF17b mRNA is seen in the postmeiotic male germ cells. ( C ) Affinity-purified anti-KIF17b recognizes TB-RBP-associated KIF17b from testis extracts. A Western blot shows protein A-agarose beads preclearing of extracts from brain (lane 1) and testis (lane 2), brain S100 cytoplasmic extract (lane 3), anti-TB-RBP immunoprecipitation of extracts from brain (lane 4) and testis (lane 5), and the enrichment of KIF17b in the TB-RBP immunoprecipitate (lane 6). ( D ) Anti-KIF17b coimmunoprecipitates TB-RBP from testis extracts. Western blot of extract immunoprecipitated with anti-KIF17b antibodies shows TB-RBP in the immunoprecipitate. Lane 1, protein A-agarose bead preclearing; lane 2, anti-KIF17b immunoprecipitate.

Article Snippet: A partial human EST clone (GenBank accession no. ) of 937 bp encoding the C terminus of a kinesin superfamily gene matching the peptide sequences derived from the MALDI-TOF mass spectrometric analysis was amplified by RT-PCR from human testis RNA (Clontech).

Techniques: Staining, Immunoprecipitation, Affinity Purification, Mass Spectrometry, Marker, Expressing, Northern Blot, Western Blot