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    Thermo Fisher superscript iii reverse transcriptase ssiiirt
    The saxE gene was required for Pst growth in leaf extracts and in planta . A. A schematic diagram of the complementary tests pinpointing the minimal region required for rescuing the growth of 25D12 mutant in plant leaf extracts. Filled arrows indicate genes annotated by the sequencing project marked with locus tags on the top. The filled triangle shows the insertion site of Ω‐Km transposon in mutant 25D12 . Genomic <t>DNA</t> fragments downstream the transposon insertion site were tested for the ability to confer tolerance to leaf extracts. B. The minimal region that protected 25D12 in leaf extracts also rescued the growth of the mutant on Arabidopsis plant. Bacterial inocula were infiltrated into Arabidopsis leaves at OD 600 = 0.001. Samples were taken at 0 and 3 dpi for colony counts. The saxE orf was required for bacterial virulence on Arabidopsis (C) and tomato (D) plants. Wild‐type Pst , Pst ▵saxE and the complemented strain Pst ▵saxE ‐ saxEhis were infiltrated into Arabidopsis (C) and tomato (D) leaves at OD 600 = 0.001. Samples were taken at 0 and 3 dpi for colony counts. All experiments were repeated at least <t>three</t> times, and similar results were observed. Data shown are means ± SD. ** indicates significant difference ( t ‐test, P
    Superscript Iii Reverse Transcriptase Ssiiirt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3765 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii reverse transcriptase reaction
    How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small <t>RNA.</t> c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of <t>three</t> independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P
    Superscript Iii Reverse Transcriptase Reaction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reverse transcriptase reaction/product/Thermo Fisher
    Average 99 stars, based on 47 article reviews
    Price from $9.99 to $1999.99
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    The saxE gene was required for Pst growth in leaf extracts and in planta . A. A schematic diagram of the complementary tests pinpointing the minimal region required for rescuing the growth of 25D12 mutant in plant leaf extracts. Filled arrows indicate genes annotated by the sequencing project marked with locus tags on the top. The filled triangle shows the insertion site of Ω‐Km transposon in mutant 25D12 . Genomic DNA fragments downstream the transposon insertion site were tested for the ability to confer tolerance to leaf extracts. B. The minimal region that protected 25D12 in leaf extracts also rescued the growth of the mutant on Arabidopsis plant. Bacterial inocula were infiltrated into Arabidopsis leaves at OD 600 = 0.001. Samples were taken at 0 and 3 dpi for colony counts. The saxE orf was required for bacterial virulence on Arabidopsis (C) and tomato (D) plants. Wild‐type Pst , Pst ▵saxE and the complemented strain Pst ▵saxE ‐ saxEhis were infiltrated into Arabidopsis (C) and tomato (D) leaves at OD 600 = 0.001. Samples were taken at 0 and 3 dpi for colony counts. All experiments were repeated at least three times, and similar results were observed. Data shown are means ± SD. ** indicates significant difference ( t ‐test, P

    Journal: Microbial Biotechnology

    Article Title: Pseudomonas bacteriocin syringacin M released upon desiccation suppresses the growth of sensitive bacteria in plant necrotic lesions

    doi: 10.1111/1751-7915.13367

    Figure Lengend Snippet: The saxE gene was required for Pst growth in leaf extracts and in planta . A. A schematic diagram of the complementary tests pinpointing the minimal region required for rescuing the growth of 25D12 mutant in plant leaf extracts. Filled arrows indicate genes annotated by the sequencing project marked with locus tags on the top. The filled triangle shows the insertion site of Ω‐Km transposon in mutant 25D12 . Genomic DNA fragments downstream the transposon insertion site were tested for the ability to confer tolerance to leaf extracts. B. The minimal region that protected 25D12 in leaf extracts also rescued the growth of the mutant on Arabidopsis plant. Bacterial inocula were infiltrated into Arabidopsis leaves at OD 600 = 0.001. Samples were taken at 0 and 3 dpi for colony counts. The saxE orf was required for bacterial virulence on Arabidopsis (C) and tomato (D) plants. Wild‐type Pst , Pst ▵saxE and the complemented strain Pst ▵saxE ‐ saxEhis were infiltrated into Arabidopsis (C) and tomato (D) leaves at OD 600 = 0.001. Samples were taken at 0 and 3 dpi for colony counts. All experiments were repeated at least three times, and similar results were observed. Data shown are means ± SD. ** indicates significant difference ( t ‐test, P

    Article Snippet: Recombinant RNase‐free DNase I (Takara, Kusatsu, Japan) and SuperScript III Reverse Transcriptase (Invitrogen) were used to remove genomic DNA and synthesize the first‐strand cDNA respectively.

    Techniques: Mutagenesis, Sequencing

    The HERV-K LTR does not possess a functional initiator element, but silencing of Sp1 and Sp3 reduces pGL3_pck30 promoter activity. (A) HERV-K transcription initiation is not mediated by an initiator element (Inr). The putative Inr wild-type sequence, TCAGTAA, was changed into a heptamer of adenines in the LTR reporter pGL3_pck30_Inr construct. Luciferase activities of pGL3_pck30 (wild type) and the pGL3_pck30_Inr mutant were determined in GH and Mel-C9 cells at 24 h posttransfection. The promoter activity of LTRpck30 was set to 100% for each cell line. The results are means ± SD for six individual experiments. (B) Immunoblot analysis of endogenous Sp1 and Sp3 protein levels in HeLa, HCl Mel 19, GH, and Mel-C9 cell lines. Fifty micrograms of cell lysate was used. Actin served as a loading control. (C) GH and Mel-C9 cells were cotransfected with pGL3_pck30 DNA and 150 ng specific siRNA or with 150 ng unrelated siRNA. % RLU are given as means ± SD for three individual transfections. The mock level was set to 100%. HERV-K LTR activity was significantly reduced by Sp1 as well as Sp3 ablation. (D) Sp1 or Sp3 knockdown reduced HERV-K promoter activity. Mel-C9 and GH cells were transfected with 150 or 300 ng of either Sp1- or Sp3-specific siRNA. Immunoblot analysis using 30 μg of protein revealed that the amounts of Sp1 and Sp3 proteins were significantly reduced by 150 ng of the respective siRNA at 48 h posttransfection. Actin was used to control for equal loading of cell lysates.

    Journal: Journal of Virology

    Article Title: Expression of the Human Endogenous Retrovirus (HERV) Group HML-2/HERV-K Does Not Depend on Canonical Promoter Elements but Is Regulated by Transcription Factors Sp1 and Sp3 ▿

    doi: 10.1128/JVI.02539-10

    Figure Lengend Snippet: The HERV-K LTR does not possess a functional initiator element, but silencing of Sp1 and Sp3 reduces pGL3_pck30 promoter activity. (A) HERV-K transcription initiation is not mediated by an initiator element (Inr). The putative Inr wild-type sequence, TCAGTAA, was changed into a heptamer of adenines in the LTR reporter pGL3_pck30_Inr construct. Luciferase activities of pGL3_pck30 (wild type) and the pGL3_pck30_Inr mutant were determined in GH and Mel-C9 cells at 24 h posttransfection. The promoter activity of LTRpck30 was set to 100% for each cell line. The results are means ± SD for six individual experiments. (B) Immunoblot analysis of endogenous Sp1 and Sp3 protein levels in HeLa, HCl Mel 19, GH, and Mel-C9 cell lines. Fifty micrograms of cell lysate was used. Actin served as a loading control. (C) GH and Mel-C9 cells were cotransfected with pGL3_pck30 DNA and 150 ng specific siRNA or with 150 ng unrelated siRNA. % RLU are given as means ± SD for three individual transfections. The mock level was set to 100%. HERV-K LTR activity was significantly reduced by Sp1 as well as Sp3 ablation. (D) Sp1 or Sp3 knockdown reduced HERV-K promoter activity. Mel-C9 and GH cells were transfected with 150 or 300 ng of either Sp1- or Sp3-specific siRNA. Immunoblot analysis using 30 μg of protein revealed that the amounts of Sp1 and Sp3 proteins were significantly reduced by 150 ng of the respective siRNA at 48 h posttransfection. Actin was used to control for equal loading of cell lysates.

    Article Snippet: The TSS and termination sites of HERV-K were identified using a GeneRacer kit with SuperScript III reverse transcriptase (RT) (Invitrogen) according to the manufacturer's instructions.

    Techniques: Functional Assay, Activity Assay, Sequencing, Construct, Luciferase, Mutagenesis, Transfection

    HERV-K LTR reporter plasmids analyzed with a dual-luciferase reporter system. GH (germ cell tumor) (A), Mel-C9 (melanoma) (B), and HCl Mel 19 (melanoma) (C) cells were transfected with pGL3_pck30, pGL3_LTR21, pGL3_basic (promoterless), or pGL3_control (SV40 promoter) DNA. (D) GH cells were also transfected with pGL3_pck30 DNA treated with the CpG dinucleotide-methylating enzyme SssI (pGL3_pck30meth). % RLU are shown as means ± standard deviations (SD) for six (A to C) or three (D) individual transfections. LTR21 was inactive in all cell lines (A to C, 4th bars), and LTRpck30 was active in GH and Mel-C9 cells (A and B, 3rd bars) but was silenced upon methylation of CpG (D, 2nd bar).

    Journal: Journal of Virology

    Article Title: Expression of the Human Endogenous Retrovirus (HERV) Group HML-2/HERV-K Does Not Depend on Canonical Promoter Elements but Is Regulated by Transcription Factors Sp1 and Sp3 ▿

    doi: 10.1128/JVI.02539-10

    Figure Lengend Snippet: HERV-K LTR reporter plasmids analyzed with a dual-luciferase reporter system. GH (germ cell tumor) (A), Mel-C9 (melanoma) (B), and HCl Mel 19 (melanoma) (C) cells were transfected with pGL3_pck30, pGL3_LTR21, pGL3_basic (promoterless), or pGL3_control (SV40 promoter) DNA. (D) GH cells were also transfected with pGL3_pck30 DNA treated with the CpG dinucleotide-methylating enzyme SssI (pGL3_pck30meth). % RLU are shown as means ± standard deviations (SD) for six (A to C) or three (D) individual transfections. LTR21 was inactive in all cell lines (A to C, 4th bars), and LTRpck30 was active in GH and Mel-C9 cells (A and B, 3rd bars) but was silenced upon methylation of CpG (D, 2nd bar).

    Article Snippet: The TSS and termination sites of HERV-K were identified using a GeneRacer kit with SuperScript III reverse transcriptase (RT) (Invitrogen) according to the manufacturer's instructions.

    Techniques: Luciferase, Transfection, Methylation

    Three GC boxes mediate transcriptional activity of the HERV-K LTR.

    Journal: Journal of Virology

    Article Title: Expression of the Human Endogenous Retrovirus (HERV) Group HML-2/HERV-K Does Not Depend on Canonical Promoter Elements but Is Regulated by Transcription Factors Sp1 and Sp3 ▿

    doi: 10.1128/JVI.02539-10

    Figure Lengend Snippet: Three GC boxes mediate transcriptional activity of the HERV-K LTR.

    Article Snippet: The TSS and termination sites of HERV-K were identified using a GeneRacer kit with SuperScript III reverse transcriptase (RT) (Invitrogen) according to the manufacturer's instructions.

    Techniques: Activity Assay

    RNA editing of EEF-derived miRNA. (A) Graphic illustration of A-to-I editing, which is catalyzed by ADAR enzymes. miRNA editing can occur in seed regions (as shown here) or outside of the seed regions. This graph was created with BioRender. (B) Boxplots showing the editing levels (median with interquartile range), in control and KA samples at the two different time points for six edited sites detected in EEF miRNAs. (C) The table shows evidence supporting the edited sites and (D) sequence motifs of the edited sites. This shows the nucleotide preferences of all sequences surrounding the six edited sites [three-bases long, with the edited nucleotide Adenosine (A) is centered]. All the six edited sites have preferable sequence motifs for ADAR enzymes, which is (1) over-represented uridine (U) but depleted guanosine (G) at the nucleotides before the edited sites and (2) over-represented guanosine at the nucleotides after the edited sites. Confidence of edited site ( Li et al., 2018 ) reported in brain ( Alon et al., 2012 ) and reported in exosomes ( Nigita et al., 2018 ).

    Journal: Frontiers in Neuroscience

    Article Title: Altered Biogenesis and MicroRNA Content of Hippocampal Exosomes Following Experimental Status Epilepticus

    doi: 10.3389/fnins.2019.01404

    Figure Lengend Snippet: RNA editing of EEF-derived miRNA. (A) Graphic illustration of A-to-I editing, which is catalyzed by ADAR enzymes. miRNA editing can occur in seed regions (as shown here) or outside of the seed regions. This graph was created with BioRender. (B) Boxplots showing the editing levels (median with interquartile range), in control and KA samples at the two different time points for six edited sites detected in EEF miRNAs. (C) The table shows evidence supporting the edited sites and (D) sequence motifs of the edited sites. This shows the nucleotide preferences of all sequences surrounding the six edited sites [three-bases long, with the edited nucleotide Adenosine (A) is centered]. All the six edited sites have preferable sequence motifs for ADAR enzymes, which is (1) over-represented uridine (U) but depleted guanosine (G) at the nucleotides before the edited sites and (2) over-represented guanosine at the nucleotides after the edited sites. Confidence of edited site ( Li et al., 2018 ) reported in brain ( Alon et al., 2012 ) and reported in exosomes ( Nigita et al., 2018 ).

    Article Snippet: For analysis of transcripts, complementary DNA (cDNA) was produced from 1 μg of the total RNA by reverse transcription using Superscript III Reverse Transcriptase enzyme (Invitrogen).

    Techniques: Derivative Assay, Sequencing

    Impact of MyD88 reactivation on the expression of factors that govern MO/DC function in infection and inflammation. (A, B) WT, MyD OFF , CD11c-MyD ON and LysM-MyD ON mice were infected orally with C . rodentium . On day 4 p.i. leukocytes were isolated from the cLP and sorted for DC (lin − CD64 − MHC-II + CD26 + CD11c + ) and MO (lin − CD64 + F4/80 + MHC-II + ). As lineage marker, antibodies against CD3, CD19, B220 and NK1.1 were included (A) Heat map displaying expression of genes involved in MNP function in infection and inflammation analyzed by real-time PCR from sorted DC/MO fractions. Data presented as mean log2 value of relative mRNA expression (see color scale). (B) Bar graphs show mean expression of selected genes in sorted DC/MO fractions relative to the expression of 5 housekeeping genes ( Actb , B2m , Gapdh , Gusb , Hsp90ab1 ). Data were pooled from three individual experiments with n = 4–6 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test; *p

    Journal: PLoS Pathogens

    Article Title: MyD88 signaling in dendritic cells and the intestinal epithelium controls immunity against intestinal infection with C. rodentium

    doi: 10.1371/journal.ppat.1006357

    Figure Lengend Snippet: Impact of MyD88 reactivation on the expression of factors that govern MO/DC function in infection and inflammation. (A, B) WT, MyD OFF , CD11c-MyD ON and LysM-MyD ON mice were infected orally with C . rodentium . On day 4 p.i. leukocytes were isolated from the cLP and sorted for DC (lin − CD64 − MHC-II + CD26 + CD11c + ) and MO (lin − CD64 + F4/80 + MHC-II + ). As lineage marker, antibodies against CD3, CD19, B220 and NK1.1 were included (A) Heat map displaying expression of genes involved in MNP function in infection and inflammation analyzed by real-time PCR from sorted DC/MO fractions. Data presented as mean log2 value of relative mRNA expression (see color scale). (B) Bar graphs show mean expression of selected genes in sorted DC/MO fractions relative to the expression of 5 housekeeping genes ( Actb , B2m , Gapdh , Gusb , Hsp90ab1 ). Data were pooled from three individual experiments with n = 4–6 mice per group. Error bar represents +SEM. One-Way ANOVA with Bonferroni’s Multiple Comparison test; *p

    Article Snippet: 50–100 ng of amplified mRNA was transcribed into cDNA using SuperScript III Reverse Transcriptase Kit (Thermo Fisher Scientific).

    Techniques: Expressing, Infection, Mouse Assay, Isolation, Marker, Real-time Polymerase Chain Reaction

    Induction of Id1-3 ( A ), endogenous expression of p21 and p53 ( B ), and expression of p53 ( C ), p21 ( D ), p27 ( E ), p107 ( F ) and p130 ( G ) upon recombinant BMP4 administration to cell cultures 24hrs after administration of 40ng/ml recombinant BMP4. Messenger RNA expression levels of Id1, Id2 and Id3 are significantly up-regulated in WERI-Rb-1 ( A ) upon a BMP4 administration, whereas no changes in p53, p21, p27, p107 and p130 expression level were detectable ( C-G ). Endogeneous levels of p21 and p53 are, however, elevated in WERI-Rb1 cells ( B ). Data were gained from real-time PCR analyses of three independent experiments. Cells that were only treated with 0.1% BSA in 4mM HCl, the solvent for recombinant human BMP4, were taken as a control (ctr.) group for A and C-G , healthy human retina was used as a reference in B and the expression level was set as 1, respectively.

    Journal: International Journal of Biological Sciences

    Article Title: Bone morphogenetic protein 4 (BMP4) signaling in retinoblastoma cells

    doi:

    Figure Lengend Snippet: Induction of Id1-3 ( A ), endogenous expression of p21 and p53 ( B ), and expression of p53 ( C ), p21 ( D ), p27 ( E ), p107 ( F ) and p130 ( G ) upon recombinant BMP4 administration to cell cultures 24hrs after administration of 40ng/ml recombinant BMP4. Messenger RNA expression levels of Id1, Id2 and Id3 are significantly up-regulated in WERI-Rb-1 ( A ) upon a BMP4 administration, whereas no changes in p53, p21, p27, p107 and p130 expression level were detectable ( C-G ). Endogeneous levels of p21 and p53 are, however, elevated in WERI-Rb1 cells ( B ). Data were gained from real-time PCR analyses of three independent experiments. Cells that were only treated with 0.1% BSA in 4mM HCl, the solvent for recombinant human BMP4, were taken as a control (ctr.) group for A and C-G , healthy human retina was used as a reference in B and the expression level was set as 1, respectively.

    Article Snippet: Equal amounts of RNA were subjected to an RT-reaction using oligo(dt)20 primers (Invitrogen), employing the SuperScript III reverse transcriptase system (Invitrogen) and following the manufacturer`s instructions.

    Techniques: Expressing, Recombinant, RNA Expression, Real-time Polymerase Chain Reaction

    RNase R treatments of dsRNA samples, where three consecutive digests were carried out, with 10 μL samples saved at each step for RT-PCR screening ( lower branch ), and a parallel no-enzyme control series ( upper branch ) to validate the procedure. Denaturation at 95 °C for 8 min was carried out before each RNase R treatment.

    Journal: Viruses

    Article Title: Blackcurrant Leaf Chlorosis Associated Virus: Evidence of the Presence of Circular RNA during Infections

    doi: 10.3390/v10050260

    Figure Lengend Snippet: RNase R treatments of dsRNA samples, where three consecutive digests were carried out, with 10 μL samples saved at each step for RT-PCR screening ( lower branch ), and a parallel no-enzyme control series ( upper branch ) to validate the procedure. Denaturation at 95 °C for 8 min was carried out before each RNase R treatment.

    Article Snippet: The RT-PCR tests were performed as two-step reactions, with the cDNA synthesis steps using SuperScript III reverse transcriptase kits, and PCR steps using Platinum Hi-Fi Taq DNA polymerase (Thermo-Fisher, Waltham, MA, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction

    Expression level of pri- OsamiR395h and its target genes in rice leaf and root tissues at different developmental stages. Total RNA samples were prepared from leaf and root tissues of rice harvested at indicated time points and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. Experiment was repeated three times.

    Journal: Scientific Reports

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    doi: 10.1038/srep28791

    Figure Lengend Snippet: Expression level of pri- OsamiR395h and its target genes in rice leaf and root tissues at different developmental stages. Total RNA samples were prepared from leaf and root tissues of rice harvested at indicated time points and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. Experiment was repeated three times.

    Article Snippet: To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    NtamiR395 and NtaSULTR2 exhibit opposite expression patterns in tobacco roots. Real-time PCR analysis of expressions of NtaSULTR2 and mature NtamiR395 under different sulfate concentrations. Total RNA samples were prepared from ( a ) leaf tissue and ( b ) root tissue of four weeks old tobacco grown in MS medium with 0, 20, 1500 or 2000 μM (NH 4+ ) 2 SO 4 . NtaL25 was used as a reference gene. Data are presented as means of three technical replicates and two biological replicates, error bars represent SD (n = 6). The statistically significant difference between groups was determined by one-way ANOVA (F(df between , df within ) = F ration, p = p-value, where df = degrees of freedom). Means not sharing the same letter are statistically significantly different (P

    Journal: Scientific Reports

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    doi: 10.1038/srep28791

    Figure Lengend Snippet: NtamiR395 and NtaSULTR2 exhibit opposite expression patterns in tobacco roots. Real-time PCR analysis of expressions of NtaSULTR2 and mature NtamiR395 under different sulfate concentrations. Total RNA samples were prepared from ( a ) leaf tissue and ( b ) root tissue of four weeks old tobacco grown in MS medium with 0, 20, 1500 or 2000 μM (NH 4+ ) 2 SO 4 . NtaL25 was used as a reference gene. Data are presented as means of three technical replicates and two biological replicates, error bars represent SD (n = 6). The statistically significant difference between groups was determined by one-way ANOVA (F(df between , df within ) = F ration, p = p-value, where df = degrees of freedom). Means not sharing the same letter are statistically significantly different (P

    Article Snippet: To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Mass Spectrometry

    Predicted target OsaSULTR1 and OsaSULTR2 exhibit opposite expression patterns to that of the OsamiR395 in rice root. ( a ) Target sites of the four putative OsamiR395 target genes in rice. The target sites were compared with the complementary sequence of mature OsamiR395h . Asterisks indicate the identical sequences. ( b ) RT-PCR analysis of expression levels of the OsamiR395 putative targets. Total RNA samples used for RT-PCR were extracted from leaf and root tissues of two weeks old rice grown in N6 medium with 0, 20, 1500 or 2000 μM (NH 4+ ) 2 SO 4 and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. Experiment was repeated three times. ( c ) Stem-loop real-time RT-PCR analysis of mature OsamiR395 and real-time RT-PCR analysis of pri-OsamiR395h . Total RNA samples were prepared from leaf and root tissues of two weeks old rice grown in regular N6 medium (+S) or N6 medium without SO 4 + (−S) and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. ( d ) Real-time RT-PCR analysis was also conducted to determine the expression levels of the OsamiR395 putative targets in rice leaves and roots. Total RNA samples were prepared as in ( c ) and used for real-time RT-PCR analysis. OsaSIZ1 was used as a reference gene. For ( c,d ), data are presented as means of two independent biological replicates and three technical replicates, error bars represent SD (n = 6). Asterisks indicate the significant differences between expression levels under −S and +S conditions. P

    Journal: Scientific Reports

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    doi: 10.1038/srep28791

    Figure Lengend Snippet: Predicted target OsaSULTR1 and OsaSULTR2 exhibit opposite expression patterns to that of the OsamiR395 in rice root. ( a ) Target sites of the four putative OsamiR395 target genes in rice. The target sites were compared with the complementary sequence of mature OsamiR395h . Asterisks indicate the identical sequences. ( b ) RT-PCR analysis of expression levels of the OsamiR395 putative targets. Total RNA samples used for RT-PCR were extracted from leaf and root tissues of two weeks old rice grown in N6 medium with 0, 20, 1500 or 2000 μM (NH 4+ ) 2 SO 4 and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. Experiment was repeated three times. ( c ) Stem-loop real-time RT-PCR analysis of mature OsamiR395 and real-time RT-PCR analysis of pri-OsamiR395h . Total RNA samples were prepared from leaf and root tissues of two weeks old rice grown in regular N6 medium (+S) or N6 medium without SO 4 + (−S) and used for RT-PCR analysis. OsaSIZ1 was used as a reference gene. ( d ) Real-time RT-PCR analysis was also conducted to determine the expression levels of the OsamiR395 putative targets in rice leaves and roots. Total RNA samples were prepared as in ( c ) and used for real-time RT-PCR analysis. OsaSIZ1 was used as a reference gene. For ( c,d ), data are presented as means of two independent biological replicates and three technical replicates, error bars represent SD (n = 6). Asterisks indicate the significant differences between expression levels under −S and +S conditions. P

    Article Snippet: To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer.

    Techniques: Expressing, Sequencing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    Heterologous expression of pri- OsamiR395h in Nicotiana tabacum . ( a ) The Schematic diagram of rice pri- OsamiR395h overexpression construct. Rice pri- OsamiR395h sequence containing stem-loop structure of OsamiR395h was cloned from rice genomic DNA and put under the control of the CaMV35S promoter. The hptII gene driven by CaMV35S promoter was used as selectable maker. The pre- OsamiR395h sequence was underlined. Sequence emphasized with red color indicates the mature miR395h . LB, Left border; RB, right border. ( b ) RT-PCR analysis of pri- OsamiR395h expression in wild type and three transgenic tobacco lines. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco plants grown in MS medium. NtaL25 was used as reference gene. ( c ) Small RNA northern blotting analysis of mature miR395 transcripts in wild type and three transgenic tobacco lines. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco plants grown in MS medium. rRNA was used as loading control. WT: wild type plant. OE: overexpression line.

    Journal: Scientific Reports

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    doi: 10.1038/srep28791

    Figure Lengend Snippet: Heterologous expression of pri- OsamiR395h in Nicotiana tabacum . ( a ) The Schematic diagram of rice pri- OsamiR395h overexpression construct. Rice pri- OsamiR395h sequence containing stem-loop structure of OsamiR395h was cloned from rice genomic DNA and put under the control of the CaMV35S promoter. The hptII gene driven by CaMV35S promoter was used as selectable maker. The pre- OsamiR395h sequence was underlined. Sequence emphasized with red color indicates the mature miR395h . LB, Left border; RB, right border. ( b ) RT-PCR analysis of pri- OsamiR395h expression in wild type and three transgenic tobacco lines. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco plants grown in MS medium. NtaL25 was used as reference gene. ( c ) Small RNA northern blotting analysis of mature miR395 transcripts in wild type and three transgenic tobacco lines. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco plants grown in MS medium. rRNA was used as loading control. WT: wild type plant. OE: overexpression line.

    Article Snippet: To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer.

    Techniques: Expressing, Over Expression, Construct, Sequencing, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Mass Spectrometry, Northern Blot

    Sulfate deficiency induces accumulation of OsamiR395 in rice. ( a ) Small RNA northern blotting analysis of mature OsamiR395 under different sulfate concentrations. Total RNA samples were prepared from leaf and root tissues of two weeks old rice grown in N6 medium with 0, 20, 1500 or 2000 μM (NH 4+ ) 2 SO 4 and used for small RNA northern blotting analysis. Antisense oligonucleotides of OsamiR395 was labeled with γ-[ 32 P]ATP and used as probe to detect the transcript level of mature OsamiR395 . rRNA was used as a loading control. ( b ) Stem-loop real-time PCR analysis of mature OsamiR395 under different sulfate concentrations. Total RNA samples were prepared as in ( a ) and used for stem-loop real-time PCR analysis. OsaSIZ1 was used as a reference gene. Data are presented as means of three technique replicates, error bars represent SD (n = 3). ( c ) Real-time PCR analysis of rice pri- OsamiR395h under different sulfate concentrations. Total RNA samples were prepared as in ( a ) and used for real-time PCR analysis. OsaSIZ1 was used as a reference gene. Data are presented as means of three technique replicates, error bars represent SD (n = 3). The statistically significant difference between groups was determined by one-way ANOVA (F(df between , df within ) = F ration, p = p-value, where df = degrees of freedom). Means not sharing the same letter are statistically significantly different (P

    Journal: Scientific Reports

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    doi: 10.1038/srep28791

    Figure Lengend Snippet: Sulfate deficiency induces accumulation of OsamiR395 in rice. ( a ) Small RNA northern blotting analysis of mature OsamiR395 under different sulfate concentrations. Total RNA samples were prepared from leaf and root tissues of two weeks old rice grown in N6 medium with 0, 20, 1500 or 2000 μM (NH 4+ ) 2 SO 4 and used for small RNA northern blotting analysis. Antisense oligonucleotides of OsamiR395 was labeled with γ-[ 32 P]ATP and used as probe to detect the transcript level of mature OsamiR395 . rRNA was used as a loading control. ( b ) Stem-loop real-time PCR analysis of mature OsamiR395 under different sulfate concentrations. Total RNA samples were prepared as in ( a ) and used for stem-loop real-time PCR analysis. OsaSIZ1 was used as a reference gene. Data are presented as means of three technique replicates, error bars represent SD (n = 3). ( c ) Real-time PCR analysis of rice pri- OsamiR395h under different sulfate concentrations. Total RNA samples were prepared as in ( a ) and used for real-time PCR analysis. OsaSIZ1 was used as a reference gene. Data are presented as means of three technique replicates, error bars represent SD (n = 3). The statistically significant difference between groups was determined by one-way ANOVA (F(df between , df within ) = F ration, p = p-value, where df = degrees of freedom). Means not sharing the same letter are statistically significantly different (P

    Article Snippet: To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer.

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

    Identification of a sulfate transporter gene, NtaSULTR2 , the target of miR395 in tobacco. ( a ) RT-PCR analysis of NtaSULTR2 expression in tobacco. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco and used for RT-PCR analysis. NtaL25 was used as a reference gene. Experiment was repeated three times. ( b ) General structure of tobacco gene NtSULTR2 . NtaSULTR2 with a length of 1335 bp contains a sulfate transporter domain between 724 bp to 1332 bp, and a miR395 target site between 135 bp to 156 bp. The target site was compared with the complementary sequence of mature OsamIR395h and NtamiR395 . Asterisks indicate the identical sequences. ( c ) phylogenetic analysis of NtaSULTR2 protein. Protein sequences of NtaSULTR2 and 16 sulfate transporters of rice and Arabidopsis were used to establish phylogenetic tree with MEGA6. In this phylogenetic tree, NtaSULTR2 protein is classified into the second group of sulfate transporter subfamily together with AthSULTR2;1, AthSULTR2;2 and OsaSULTR2;1.

    Journal: Scientific Reports

    Article Title: Heterologous expression of a rice miR395 gene in Nicotiana tabacum impairs sulfate homeostasis

    doi: 10.1038/srep28791

    Figure Lengend Snippet: Identification of a sulfate transporter gene, NtaSULTR2 , the target of miR395 in tobacco. ( a ) RT-PCR analysis of NtaSULTR2 expression in tobacco. Total RNA samples were prepared from two weeks old wild type and transgenic tobacco and used for RT-PCR analysis. NtaL25 was used as a reference gene. Experiment was repeated three times. ( b ) General structure of tobacco gene NtSULTR2 . NtaSULTR2 with a length of 1335 bp contains a sulfate transporter domain between 724 bp to 1332 bp, and a miR395 target site between 135 bp to 156 bp. The target site was compared with the complementary sequence of mature OsamIR395h and NtamiR395 . Asterisks indicate the identical sequences. ( c ) phylogenetic analysis of NtaSULTR2 protein. Protein sequences of NtaSULTR2 and 16 sulfate transporters of rice and Arabidopsis were used to establish phylogenetic tree with MEGA6. In this phylogenetic tree, NtaSULTR2 protein is classified into the second group of sulfate transporter subfamily together with AthSULTR2;1, AthSULTR2;2 and OsaSULTR2;1.

    Article Snippet: To determine the transcript level of mature miR395 , the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)20 was replaced with miR395 specific reverse transcription primer.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Sequencing

    BMMSCs express Tet1, Tet2, and Tet3. a , b Both human (h) and mouse (m) BMMSCs expressed Tet1, Tet2, and Tet3, as assessed by western blotting ( a ) and qPCR ( b ). c Immunocytofluorescent staining showed that CD146-positive BMMSCs expressed Tet1, Tet2, and Tet3. Scale bar, 50 μm. Results are from three independent experiments. *** p

    Journal: Nature Communications

    Article Title: Tet1 and Tet2 maintain mesenchymal stem cell homeostasis via demethylation of the P2rX7 promoter

    doi: 10.1038/s41467-018-04464-6

    Figure Lengend Snippet: BMMSCs express Tet1, Tet2, and Tet3. a , b Both human (h) and mouse (m) BMMSCs expressed Tet1, Tet2, and Tet3, as assessed by western blotting ( a ) and qPCR ( b ). c Immunocytofluorescent staining showed that CD146-positive BMMSCs expressed Tet1, Tet2, and Tet3. Scale bar, 50 μm. Results are from three independent experiments. *** p

    Article Snippet: For qPCR of mRNA, we used SuperScript III Reverse Transcriptase (RT) kit (Invitrogen) to prepare the complementary DNA (cDNA). qPCR was performed using SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and gene-specific primers.

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Staining

    SA-miRNAs from oncogenic MIR17HG and tumor-suppressive MIR100HG clusters function to establish senescent phenotype in hADSCs. a Percentage of SA-β-Gal positive cells among the total amount of cells counted after transient transfection of the mimics of the SA-miRNAs from either the MIR17HG ( mir-17-5p , mir-18a-5p , mir-19a-3p , mir-20a-5p and mir-92a1-5p ) or the MIR100HG ( mir-125b1-5p , mir-1let7a-2-3p , mir-100-5p ) clusters separately or after simultaneous transfection by a full set of the SA-miRNA mimics from both clusters in SR hADSCs. SA-miRNA mimics were transfected with FITC-labeled control to account for the transfection efficiency as described in material and methods and electronic supplementary materials . The transfection efficiency for each combination is shown in (Supplementary Figure S8D ) and is expressed as a percentage of green cells among the total DAPI-positive cells ( n ) counted under the fluorescent microscope. b Quantitation of the BrdU incorporation in SA-miRNA mimics-transfected SR hADSCs (FITC+) 72 hrs post-transfection presented as FITC+ BrdU+ ( red bar ). Quantification of cells positive for SEN-associated markers P21 WAF1/Cip1 and γH2AX persistent DDR focal staining is performed under the same conditions in FITC+ ( red bars ) and FITC− cells. Results were plotted on the graphs as the averages of three independent experiments (biological replicates n = 3) with the standard deviation of data. Cell counted in each experiments: BrdU staining n 1 = 440, n 2 = 279, n 3 = 334; γH2AX staining n 1 = 117, n 2 = 109, n 3 = 133; P21 WAF1/Cip1 staining n 1 = 118, n 2 = 135, n 3 = 90. P -values (p) were calculated as Students’ two-tailed test: BrdU labeling experiment ***p

    Journal: NPJ Aging and Mechanisms of Disease

    Article Title: Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells

    doi: 10.1038/s41514-017-0006-y

    Figure Lengend Snippet: SA-miRNAs from oncogenic MIR17HG and tumor-suppressive MIR100HG clusters function to establish senescent phenotype in hADSCs. a Percentage of SA-β-Gal positive cells among the total amount of cells counted after transient transfection of the mimics of the SA-miRNAs from either the MIR17HG ( mir-17-5p , mir-18a-5p , mir-19a-3p , mir-20a-5p and mir-92a1-5p ) or the MIR100HG ( mir-125b1-5p , mir-1let7a-2-3p , mir-100-5p ) clusters separately or after simultaneous transfection by a full set of the SA-miRNA mimics from both clusters in SR hADSCs. SA-miRNA mimics were transfected with FITC-labeled control to account for the transfection efficiency as described in material and methods and electronic supplementary materials . The transfection efficiency for each combination is shown in (Supplementary Figure S8D ) and is expressed as a percentage of green cells among the total DAPI-positive cells ( n ) counted under the fluorescent microscope. b Quantitation of the BrdU incorporation in SA-miRNA mimics-transfected SR hADSCs (FITC+) 72 hrs post-transfection presented as FITC+ BrdU+ ( red bar ). Quantification of cells positive for SEN-associated markers P21 WAF1/Cip1 and γH2AX persistent DDR focal staining is performed under the same conditions in FITC+ ( red bars ) and FITC− cells. Results were plotted on the graphs as the averages of three independent experiments (biological replicates n = 3) with the standard deviation of data. Cell counted in each experiments: BrdU staining n 1 = 440, n 2 = 279, n 3 = 334; γH2AX staining n 1 = 117, n 2 = 109, n 3 = 133; P21 WAF1/Cip1 staining n 1 = 118, n 2 = 135, n 3 = 90. P -values (p) were calculated as Students’ two-tailed test: BrdU labeling experiment ***p

    Article Snippet: Total RNA and miRNA was reverse transcribed by using the high capacity superscript III reverse transcriptase (Life Technologies) and the Mystic miRNA cDNA synthesis Mix kit (Sigma-Aldrich), respectively.

    Techniques: Transfection, Labeling, Microscopy, Quantitation Assay, BrdU Incorporation Assay, Staining, Standard Deviation, BrdU Staining, Two Tailed Test

    MiRNA clusters and SEN-associated miRNA (SA-miRNAs) discovered via RNA-seq analysis and experimentally validated by qPCR. a Differential expression of non-coding RNA genes in SR versus SEN hADSCs revealed by RNA-seq analysis. Fold-change values (log 2 SEN/SR) are shown on the x -axis and RPKM differences (log 2 SEN-SR) are shown on the y -axis. SEN upregulated non-coding RNA genes are shown in red (upper right quadrant). b Genomic locations and locus names for SEN upregulated miRNA gene clusters revealed by RNA-seq analysis. c Graphical representation of oncogenic MIR17HG locus and qPCR analysis of mature mirRNA expression in SR ( blue bar ) and senescent (SEN, red bar ) states of hADSCs. Relative expression of either passenger strand mature miRNAs (depicted in the graphs as -3p ) or guide strand mature miRNAs (depicted in the graphs as -5p ) to U6 small RNA was measured. Data are shown as fold change (ΔΔC τ ) The mean ± SD from three independent experiments is shown. The statistical difference was evaluated by Student’s t- test and P- value ( p ) related to experimental measurements and are listed under the graphs, where *** p

    Journal: NPJ Aging and Mechanisms of Disease

    Article Title: Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells

    doi: 10.1038/s41514-017-0006-y

    Figure Lengend Snippet: MiRNA clusters and SEN-associated miRNA (SA-miRNAs) discovered via RNA-seq analysis and experimentally validated by qPCR. a Differential expression of non-coding RNA genes in SR versus SEN hADSCs revealed by RNA-seq analysis. Fold-change values (log 2 SEN/SR) are shown on the x -axis and RPKM differences (log 2 SEN-SR) are shown on the y -axis. SEN upregulated non-coding RNA genes are shown in red (upper right quadrant). b Genomic locations and locus names for SEN upregulated miRNA gene clusters revealed by RNA-seq analysis. c Graphical representation of oncogenic MIR17HG locus and qPCR analysis of mature mirRNA expression in SR ( blue bar ) and senescent (SEN, red bar ) states of hADSCs. Relative expression of either passenger strand mature miRNAs (depicted in the graphs as -3p ) or guide strand mature miRNAs (depicted in the graphs as -5p ) to U6 small RNA was measured. Data are shown as fold change (ΔΔC τ ) The mean ± SD from three independent experiments is shown. The statistical difference was evaluated by Student’s t- test and P- value ( p ) related to experimental measurements and are listed under the graphs, where *** p

    Article Snippet: Total RNA and miRNA was reverse transcribed by using the high capacity superscript III reverse transcriptase (Life Technologies) and the Mystic miRNA cDNA synthesis Mix kit (Sigma-Aldrich), respectively.

    Techniques: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Expressing

    Differential expression of mRNA and protein targets of SA-miRNAs. a Schematic showing the mRNA degradation versus translational repression modes of miRNA regulation. b Differential expression of protein-coding mRNAs in SR versus SEN hADSCs. Fold-change values (log 2 SEN/SR) are shown on the x -axis, and RPKM differences (log 2 SEN-SR) are shown on the y -axis. SEN downregulated protein-coding mRNAs are shown in green (lower left quadrant). c Heatmap depicting differential expression of proteins in SR versus SEN hADSCs. Normalized protein expression levels are shown for three replicate samples each of SR versus SEN hADSCs (see z-score color scale). SEN downregulated proteins are shown in the lower right quadrant. d A flowchart illustrating the approach to identifying downregulated SA-miRNA targets is shown along with a Venn-diagram indicating the numbers of genes or proteins identified via each method and the numbers identified by multiple methods

    Journal: NPJ Aging and Mechanisms of Disease

    Article Title: Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells

    doi: 10.1038/s41514-017-0006-y

    Figure Lengend Snippet: Differential expression of mRNA and protein targets of SA-miRNAs. a Schematic showing the mRNA degradation versus translational repression modes of miRNA regulation. b Differential expression of protein-coding mRNAs in SR versus SEN hADSCs. Fold-change values (log 2 SEN/SR) are shown on the x -axis, and RPKM differences (log 2 SEN-SR) are shown on the y -axis. SEN downregulated protein-coding mRNAs are shown in green (lower left quadrant). c Heatmap depicting differential expression of proteins in SR versus SEN hADSCs. Normalized protein expression levels are shown for three replicate samples each of SR versus SEN hADSCs (see z-score color scale). SEN downregulated proteins are shown in the lower right quadrant. d A flowchart illustrating the approach to identifying downregulated SA-miRNA targets is shown along with a Venn-diagram indicating the numbers of genes or proteins identified via each method and the numbers identified by multiple methods

    Article Snippet: Total RNA and miRNA was reverse transcribed by using the high capacity superscript III reverse transcriptase (Life Technologies) and the Mystic miRNA cDNA synthesis Mix kit (Sigma-Aldrich), respectively.

    Techniques: Expressing

    Viral loads and plasma levels of IFNα, IL-15, IL-18 and IL-1βin sample time courses from three subjects acutely infected with HIV. A is a US plasma donor, whose sample time course is plotted in days and is aligned relative to the time (designated day 0) when the plasma viral load first reached 100 copies/ml (i.e. the start of the viral expansion phase). B and C are CHAVI 001 subjects, whose sample time courses are plotted in weeks, and are aligned relative to the time of study enrollment (week 0). Subject B started ART just after study enrollment, as indicated by the black arrow. Viral load data is plotted as open squares joined by dotted lines, and is expressed as log 10 RNA copies/ml. Data for each cytokine is plotted as filled symbols joined by solid lines, and is expressed as pg/ml.

    Journal: PLoS ONE

    Article Title: Cross-Sectional Detection of Acute HIV Infection: Timing of Transmission, Inflammation and Antiretroviral Therapy

    doi: 10.1371/journal.pone.0019617

    Figure Lengend Snippet: Viral loads and plasma levels of IFNα, IL-15, IL-18 and IL-1βin sample time courses from three subjects acutely infected with HIV. A is a US plasma donor, whose sample time course is plotted in days and is aligned relative to the time (designated day 0) when the plasma viral load first reached 100 copies/ml (i.e. the start of the viral expansion phase). B and C are CHAVI 001 subjects, whose sample time courses are plotted in weeks, and are aligned relative to the time of study enrollment (week 0). Subject B started ART just after study enrollment, as indicated by the black arrow. Viral load data is plotted as open squares joined by dotted lines, and is expressed as log 10 RNA copies/ml. Data for each cytokine is plotted as filled symbols joined by solid lines, and is expressed as pg/ml.

    Article Snippet: HIV-1 RNA was reverse transcribed to cDNA using Superscript III Reverse Transcriptase (RT) System (Invitrogen.)

    Techniques: Infection

    A Second Layer of Cooperativity Reinforces the Folded Modules (A) Contacts of P5c with the 3WJ and the MC in the native, “packed” conformation. Contacts are shown as dashed lines in the secondary structure diagram (left) and as magenta lines in the three-dimensional structure image (right). (B) Substitution of U168 to C is expected to perturb the long-range non-canonical base pair of U168 with G188, weakening the packing transition of P5c. (C) SHAPE footprinting of mutants that weaken or knock out the P5c packing transition. Results are shown for U168C (B) and for the nat+3v1 mutant, which stabilizes the P5c native secondary structure but eliminates the long-range interaction with the MC by extending the P5c helix ( Figure 4A ). MC formation occurs for both mutants, as reflected by protection of A186 (open symbols) and partial protection of A184 (closed symbols), and P5c packing is detected only for the wild-type P5abc RNA, as reflected in a further protection of A184. (D) Construct designed to enforce the native P5c secondary structure (C165A/G175U, designated A:U) and knock out MC formation (A186U). (E) DMS footprinting of the P5abc mutant shown in (D). P5c packing with the 3WJ is monitored by enhanced DMS reactivity of A173 ( Supplemental Experimental Procedures ). See also Figure S6 .

    Journal: Cell reports

    Article Title: Hidden Structural Modules in a Cooperative RNA Folding Transition

    doi: 10.1016/j.celrep.2018.02.101

    Figure Lengend Snippet: A Second Layer of Cooperativity Reinforces the Folded Modules (A) Contacts of P5c with the 3WJ and the MC in the native, “packed” conformation. Contacts are shown as dashed lines in the secondary structure diagram (left) and as magenta lines in the three-dimensional structure image (right). (B) Substitution of U168 to C is expected to perturb the long-range non-canonical base pair of U168 with G188, weakening the packing transition of P5c. (C) SHAPE footprinting of mutants that weaken or knock out the P5c packing transition. Results are shown for U168C (B) and for the nat+3v1 mutant, which stabilizes the P5c native secondary structure but eliminates the long-range interaction with the MC by extending the P5c helix ( Figure 4A ). MC formation occurs for both mutants, as reflected by protection of A186 (open symbols) and partial protection of A184 (closed symbols), and P5c packing is detected only for the wild-type P5abc RNA, as reflected in a further protection of A184. (D) Construct designed to enforce the native P5c secondary structure (C165A/G175U, designated A:U) and knock out MC formation (A186U). (E) DMS footprinting of the P5abc mutant shown in (D). P5c packing with the 3WJ is monitored by enhanced DMS reactivity of A173 ( Supplemental Experimental Procedures ). See also Figure S6 .

    Article Snippet: To purify modified RNA, 10 μL of the primer-bead mixture was incubated with RNA for 10 min, washed twice with 200 μL of 70% ethanol, and left to dry at room temperature for 20 min. RNA was reverse transcribed with 5 μL of Superscript III reverse transcriptase mix (Life Technologies) at 42°C for 60 min, followed by RNA degradation with 0.2 M NaOH at 90°C for 3 min.

    Techniques: Footprinting, Knock-Out, Mutagenesis, Construct

    How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small RNA. c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of three independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P

    Journal: Genome Biology

    Article Title: Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency

    doi: 10.1186/s13059-015-0846-3

    Figure Lengend Snippet: How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b . The raw data are shown in Figure S10 in Additional file 1 . Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small RNA. c In vitro transcribed sgRNA formed dimers ( upper panel ), which can be transformed into monomers by a heating and quick cooling step ( lower panel ). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b . The raw data are shown in Figure S11 in Additional file 1 . The graphs represent biological repeats from one of three independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P

    Article Snippet: One microgram of extracted RNA was reverse transcribed with SuperScript® III Reverse Transcriptase reaction (Life Technology, catalog #18080-051), according to the manufacturer’s instructions.

    Techniques: Knock-Out, Mutagenesis, Real-time Polymerase Chain Reaction, Expressing, In Vitro, Transformation Assay, Standard Deviation

    Knockout efficiency can be increased by extending the duplex and disrupting the continuous sequence of Ts. a The duplex extension. Green indicates the 3’ 34 nucleotides, which are not required for sgRNA functionality in vitro but are required in cells; red indicates the extended base pairs. b Extension of the duplex increased knockout efficiency. Constructs harboring sgRNAs targeting the CCR5 gene were co-transfected with a Cas9-expressing plasmid into TZM-bl cells. An sgRNA targeting the HIV genome served as mock control. The GFP-positive cells were sorted out 48 hours after transfection, and the gene modification rates were determined at the protein and DNA levels, respectively. Protein level disruption: the expression of CCR5 was determined by flow cytometry analysis. The raw data are shown in Figure S2 in Additional file 1 . DNA level modification rate: the genomic DNA was extracted, and the target sites were amplified and deep-sequenced with a MiSeq sequencer. The raw data are provided in Additional file 2 . c The experiment in ( b ) at the protein level was repeated for another sgRNA, sp2. The difference with ( b ) is that the cells were not sorted, but the CCR5 disruption rate was measured in GFP-positive cells. The raw data are shown in Figure S2 in Additional file 1 . d Mutation of the RNA polymerase ( Pol III ) pause signal significantly increased knockout efficiency. The mutated nucleotides are shown in bold . The raw data are shown in Figure S3 in Additional file 1 . The graphs represent biological repeats from one of three independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P

    Journal: Genome Biology

    Article Title: Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency

    doi: 10.1186/s13059-015-0846-3

    Figure Lengend Snippet: Knockout efficiency can be increased by extending the duplex and disrupting the continuous sequence of Ts. a The duplex extension. Green indicates the 3’ 34 nucleotides, which are not required for sgRNA functionality in vitro but are required in cells; red indicates the extended base pairs. b Extension of the duplex increased knockout efficiency. Constructs harboring sgRNAs targeting the CCR5 gene were co-transfected with a Cas9-expressing plasmid into TZM-bl cells. An sgRNA targeting the HIV genome served as mock control. The GFP-positive cells were sorted out 48 hours after transfection, and the gene modification rates were determined at the protein and DNA levels, respectively. Protein level disruption: the expression of CCR5 was determined by flow cytometry analysis. The raw data are shown in Figure S2 in Additional file 1 . DNA level modification rate: the genomic DNA was extracted, and the target sites were amplified and deep-sequenced with a MiSeq sequencer. The raw data are provided in Additional file 2 . c The experiment in ( b ) at the protein level was repeated for another sgRNA, sp2. The difference with ( b ) is that the cells were not sorted, but the CCR5 disruption rate was measured in GFP-positive cells. The raw data are shown in Figure S2 in Additional file 1 . d Mutation of the RNA polymerase ( Pol III ) pause signal significantly increased knockout efficiency. The mutated nucleotides are shown in bold . The raw data are shown in Figure S3 in Additional file 1 . The graphs represent biological repeats from one of three independent experiments with similar results, shown as mean ± standard deviation ( n = 3). Significance was calculated using Student's t -test: * P

    Article Snippet: One microgram of extracted RNA was reverse transcribed with SuperScript® III Reverse Transcriptase reaction (Life Technology, catalog #18080-051), according to the manufacturer’s instructions.

    Techniques: Knock-Out, Sequencing, In Vitro, Construct, Transfection, Expressing, Plasmid Preparation, Modification, Flow Cytometry, Cytometry, Amplification, Mutagenesis, Standard Deviation