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    Thermo Fisher superscript iii reverse transcriptase ssiiirt
    VTRNA2-1 is transcribed by <t>RNA</t> pol <t>III</t> and the transcript is ∼ 100 nt long. (A) VTRNA2-1 is transcribed by RNA pol III. T24 cells were treated with α-amanitin (RNA pol II inhibitor) and harvested at 24 and 48 hours after treatment. vtRNA2-1
    Superscript Iii Reverse Transcriptase Ssiiirt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4680 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
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    VTRNA2-1 is transcribed by RNA pol III and the transcript is ∼ 100 nt long. (A) VTRNA2-1 is transcribed by RNA pol III. T24 cells were treated with α-amanitin (RNA pol II inhibitor) and harvested at 24 and 48 hours after treatment. vtRNA2-1

    Journal: Blood

    Article Title: Allelic methylation levels of the noncoding VTRNA2-1 located on chromosome 5q31.1 predict outcome in AML

    doi: 10.1182/blood-2011-06-362541

    Figure Lengend Snippet: VTRNA2-1 is transcribed by RNA pol III and the transcript is ∼ 100 nt long. (A) VTRNA2-1 is transcribed by RNA pol III. T24 cells were treated with α-amanitin (RNA pol II inhibitor) and harvested at 24 and 48 hours after treatment. vtRNA2-1

    Article Snippet: RNA was RT using SuperScript III Reverse Transcriptase (Invitrogen) and random hexamers (Promega).

    Techniques:

    Trans-activation of the endogenous Abcc6 gene in TIB-73 cells Hepatocytes were transfected with a vector expressing the HNF1 α , HNF4 α or p45-NFE2 cDNAs. As a negative control, the cells were transfected with an empty vector. The levels of the endogenous mAbcc6 gene were determined by quantitative PCR with TaqMan probes specific to the murine mAbcc6 cDNA. The results were normalized to the transfection efficiency by measuring the level of expression of the neomycin-resistance gene present on the vector expressing the transcription factors. The assays were performed three times at least in triplicate. Standard errors are indicated.

    Journal: Biochimica et biophysica acta

    Article Title: HNF4? and NF-E2 are key transcriptional regulators of the murine Abcc6 gene expression

    doi: 10.1016/j.bbaexp.2006.08.002

    Figure Lengend Snippet: Trans-activation of the endogenous Abcc6 gene in TIB-73 cells Hepatocytes were transfected with a vector expressing the HNF1 α , HNF4 α or p45-NFE2 cDNAs. As a negative control, the cells were transfected with an empty vector. The levels of the endogenous mAbcc6 gene were determined by quantitative PCR with TaqMan probes specific to the murine mAbcc6 cDNA. The results were normalized to the transfection efficiency by measuring the level of expression of the neomycin-resistance gene present on the vector expressing the transcription factors. The assays were performed three times at least in triplicate. Standard errors are indicated.

    Article Snippet: Total RNA isolated (1μg) from mouse liver was reversely transcribed with the Superscript III reverse transcriptase kit primed with Oligo(dT) (Invitrogen). cDNAs were then PCR-amplified with the specific primers (see ), using the PCR Platinum Supermix (Invitrogen).

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Negative Control, Real-time Polymerase Chain Reaction

    Transcriptional activity of the mAbcc6 gene promoter Six reporter constructs were derived from the pGL3 vector and contained fragment of various length of the murine Abcc6 promoter (−2629, −1640, −928, −648, −350 and −152 to +162bp from the transcription start site). These constructs were transfected into fibroblast (NIH/3T3), hepatoma (Hepa1-6) and kidney cells (RAG). These cell lines were transfected with 1μg of plasmid and 0.1 μg of pRL-SV40 plasmid for control purposes and cultured for 24h. Luciferase activity was normalized to the renilla luciferase activity. Each value represents the mean ± standard deviation of at least three independent transfection experiments, each performed in triplicate. The luciferase activities are represented as a percentage of the −2926/+162bp promoter construct activity obtained in hepatoma (Hepa1-6).

    Journal: Biochimica et biophysica acta

    Article Title: HNF4? and NF-E2 are key transcriptional regulators of the murine Abcc6 gene expression

    doi: 10.1016/j.bbaexp.2006.08.002

    Figure Lengend Snippet: Transcriptional activity of the mAbcc6 gene promoter Six reporter constructs were derived from the pGL3 vector and contained fragment of various length of the murine Abcc6 promoter (−2629, −1640, −928, −648, −350 and −152 to +162bp from the transcription start site). These constructs were transfected into fibroblast (NIH/3T3), hepatoma (Hepa1-6) and kidney cells (RAG). These cell lines were transfected with 1μg of plasmid and 0.1 μg of pRL-SV40 plasmid for control purposes and cultured for 24h. Luciferase activity was normalized to the renilla luciferase activity. Each value represents the mean ± standard deviation of at least three independent transfection experiments, each performed in triplicate. The luciferase activities are represented as a percentage of the −2926/+162bp promoter construct activity obtained in hepatoma (Hepa1-6).

    Article Snippet: Total RNA isolated (1μg) from mouse liver was reversely transcribed with the Superscript III reverse transcriptase kit primed with Oligo(dT) (Invitrogen). cDNAs were then PCR-amplified with the specific primers (see ), using the PCR Platinum Supermix (Invitrogen).

    Techniques: Activity Assay, Construct, Derivative Assay, Plasmid Preparation, Transfection, Cell Culture, Luciferase, Standard Deviation

    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

    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

    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

    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

    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

    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

    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

    Increased alternative splicing during DR in C. elegans . a AS events in day 3 eat-2(ad1116) (shown as eat-2(-) ) worms, as exhibited by the differential usage of cassette exons (CE; dark blue bars ) or by intron retention (IR; dark brown bars ). Total spliced-in and spliced-out events in eat-2(ad1116) are shown. The genes that these events correspond to are indicated in the lighter shades . Statistical test: Irwin–Hall P value summarization was used on each individual splicing event (details in the Methods section). Four biological replicates were used for analysis. b Distribution of the number of AS events per gene during DR. Both CE and IR events are considered together. c Upper panels showing representative images of AS involving intron retention. For this, two terms were considered, reads mapping to the introns and that of the skipping junction when the intron is not included, while ensuring that flanking exons are expressed. Each blue or red box represents four biological replicates of the RNA-seq. In case of Introns 33707 and 55131, the read counts mapping to the intron or the junction were reciprocal in eat-2(ad1116) vs. WT. Lower panels : The above intron retention events belonging to m02e1.1 and y57g11c.22 are validated by RT-PCR. Two other AS events belonging to c08f11.5 and f54f3.3 are also validated below. In case of m02e1.1 , y57g11c.22 and c08f11.5 , the isoforms retaining the intron increase in eat-2(ad1116) , whereas the reverse happens in f54f3.3 . The two lanes for each sample are biological replicates. Yellow arrows indicate two alternatively spliced isoforms that show differential expression in WT and eat-2(ad1116) . Similar results were observed in at least three biological replicates. cpm counts per million. d Heat map depicting differential AS of top 42 genes in eat-2(ad1116) . J1 and J2 indicate two junctions of the same gene showing highest significant differences in their splicing indices. Statistical analysis was performed using two-tailed unpaired t- test with Sidak–Bonferroni correction for individual event. Data from four biological replicates. e KEGG pathway, Gene Ontology (GO) term enrichment and protein domains were determined by DAVID Functional Annotation tool using differentially AS genes. The numbers within the blue bars represent genes detected in the category. The listed pathways have a P value ≤ 0.05, Fisher Exact Test

    Journal: Nature Communications

    Article Title: Differential alternative splicing coupled to nonsense-mediated decay of mRNA ensures dietary restriction-induced longevity

    doi: 10.1038/s41467-017-00370-5

    Figure Lengend Snippet: Increased alternative splicing during DR in C. elegans . a AS events in day 3 eat-2(ad1116) (shown as eat-2(-) ) worms, as exhibited by the differential usage of cassette exons (CE; dark blue bars ) or by intron retention (IR; dark brown bars ). Total spliced-in and spliced-out events in eat-2(ad1116) are shown. The genes that these events correspond to are indicated in the lighter shades . Statistical test: Irwin–Hall P value summarization was used on each individual splicing event (details in the Methods section). Four biological replicates were used for analysis. b Distribution of the number of AS events per gene during DR. Both CE and IR events are considered together. c Upper panels showing representative images of AS involving intron retention. For this, two terms were considered, reads mapping to the introns and that of the skipping junction when the intron is not included, while ensuring that flanking exons are expressed. Each blue or red box represents four biological replicates of the RNA-seq. In case of Introns 33707 and 55131, the read counts mapping to the intron or the junction were reciprocal in eat-2(ad1116) vs. WT. Lower panels : The above intron retention events belonging to m02e1.1 and y57g11c.22 are validated by RT-PCR. Two other AS events belonging to c08f11.5 and f54f3.3 are also validated below. In case of m02e1.1 , y57g11c.22 and c08f11.5 , the isoforms retaining the intron increase in eat-2(ad1116) , whereas the reverse happens in f54f3.3 . The two lanes for each sample are biological replicates. Yellow arrows indicate two alternatively spliced isoforms that show differential expression in WT and eat-2(ad1116) . Similar results were observed in at least three biological replicates. cpm counts per million. d Heat map depicting differential AS of top 42 genes in eat-2(ad1116) . J1 and J2 indicate two junctions of the same gene showing highest significant differences in their splicing indices. Statistical analysis was performed using two-tailed unpaired t- test with Sidak–Bonferroni correction for individual event. Data from four biological replicates. e KEGG pathway, Gene Ontology (GO) term enrichment and protein domains were determined by DAVID Functional Annotation tool using differentially AS genes. The numbers within the blue bars represent genes detected in the category. The listed pathways have a P value ≤ 0.05, Fisher Exact Test

    Article Snippet: The quality of the ribosomal 28S and 18S on an agarose gel was used as a measure of integrity. cDNA was synthesized using 2.5 μg RNA employing Superscript III Reverse Transcriptase (Invitrogen).

    Techniques: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Two Tailed Test, Functional Assay

    Nek5 loss leads to premature centrosome separation in interphase. (a) RT-PCR of Nek5 and GAPDH with RNA collected after 72 h of siRNA treatment of U2OS cells with mock, GAPDH, or Nek5 siRNAs. Three individual Nek5 siRNAs were tested. (b) Staining of Nek5 and γ-tubulin in U2OS cells transfected with Nek5- or GL2-specific siRNAs for 48 h. (c) Quantification of cells treated as in b with centrosomes split by > 2 µm. (d) Staining of γ-tubulin and DNA in U2OS cells transfected for 48 h with GFP, GFP-Nek5WT, or GFP-Nek5KD. (e) Quantification of cells with centrosomes separated by > 2 µm in cells treated as in d. (f) U2OS cells were transfected with either siRNAs against Nek5 or GL2, or mock treated for 24 h before GFP-Nek5WT and GFP-Nek5KD RNAi sensitive and resistant constructs were transfected into the cells. Cells were harvested 24 h after plasmid transfection and analyzed by Western blotting with Nek5, GFP, and α-tubulin antibodies. (g) Quantification of cells with centrosomes separated by > 2 µm in U2OS cells treated as in f. (h) Box and whisker plot of the distance between centrosomes in mock-, GL2-, or Nek5-depleted cells, and either untransfected cells or those expressing GFP, GFP-Nek5WT, GFP-Nek5KD, or GFP-Nek2WT. Bars: (main images) 10 µm; (insets) 1 µm. *, P

    Journal: The Journal of Cell Biology

    Article Title: Nek5 promotes centrosome integrity in interphase and loss of centrosome cohesion in mitosis

    doi: 10.1083/jcb.201412099

    Figure Lengend Snippet: Nek5 loss leads to premature centrosome separation in interphase. (a) RT-PCR of Nek5 and GAPDH with RNA collected after 72 h of siRNA treatment of U2OS cells with mock, GAPDH, or Nek5 siRNAs. Three individual Nek5 siRNAs were tested. (b) Staining of Nek5 and γ-tubulin in U2OS cells transfected with Nek5- or GL2-specific siRNAs for 48 h. (c) Quantification of cells treated as in b with centrosomes split by > 2 µm. (d) Staining of γ-tubulin and DNA in U2OS cells transfected for 48 h with GFP, GFP-Nek5WT, or GFP-Nek5KD. (e) Quantification of cells with centrosomes separated by > 2 µm in cells treated as in d. (f) U2OS cells were transfected with either siRNAs against Nek5 or GL2, or mock treated for 24 h before GFP-Nek5WT and GFP-Nek5KD RNAi sensitive and resistant constructs were transfected into the cells. Cells were harvested 24 h after plasmid transfection and analyzed by Western blotting with Nek5, GFP, and α-tubulin antibodies. (g) Quantification of cells with centrosomes separated by > 2 µm in U2OS cells treated as in f. (h) Box and whisker plot of the distance between centrosomes in mock-, GL2-, or Nek5-depleted cells, and either untransfected cells or those expressing GFP, GFP-Nek5WT, GFP-Nek5KD, or GFP-Nek2WT. Bars: (main images) 10 µm; (insets) 1 µm. *, P

    Article Snippet: 1 (GAPDH, actin) or 5 µg (Nek5) of total RNA was then used for first strand cDNA synthesis with Superscript III reverse transcription (Invitrogen) according to the manufacturer’s instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Staining, Transfection, Construct, Plasmid Preparation, Western Blot, Whisker Assay, Expressing

    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