ez-rna isolation kit Search Results


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  • 96
    Zymo Research rna isolation kits
    Rna Isolation Kits, supplied by Zymo Research, used in various techniques. Bioz Stars score: 96/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nuclei ez lysis buffer
    Nuclei Ez Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 223 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biological Industries Inc ez rna isolation kit
    Expression of EHDs in CEDNIK fibroblasts. (A) <t>RNA</t> was isolated from control (SV80) and CEDNIK (F110T and F165T) fibroblasts and was subjected to Ehd1 specific quantitative real-time <t>PCR</t> (qRT-PCR). Expression level of Ehd1 in SV80 was defined as 100%. (B) Protein lysates prepared from CEDNIK (F165T, F110T) and control (SV80) fibroblasts were subjected to Western blot analysis and interacted with anti-EHD1 and anti-ERK antibodies, as a loading control. (C) Fibroblasts (SV80 and F110T) were transfected with GFP-EHD1, fixed and visualized by confocal microscopy. (D) RNA was isolated from SV80 and CEDNIK fibroblasts and subjected to Ehd2, Ehd3 or Ehd4 specific RT-PCR as detailed under Materials and Methods . (E) Control (SV80) and CEDNIK (F110T) fibroblasts were transfected with YFP-EHD2, GFP-EHD3 and YFP-EHD4 expressing plasmids for 18 h, after which they were fixed and visualized by confocal microscopy. No change in EHD1 RNA level, protein level or localization was observed in CEDNIK cells, compared to control cells. Also, no change in the intracellular localization or RNA levels of other EHDs was observed in CEDNIK cells. M- 1 kb marker. Bar,10 µm.
    Ez Rna Isolation Kit, supplied by Biological Industries Inc, used in various techniques. Bioz Stars score: 88/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio Basic Canada ez 10 rna isolation kit
    Expression of EHDs in CEDNIK fibroblasts. (A) <t>RNA</t> was isolated from control (SV80) and CEDNIK (F110T and F165T) fibroblasts and was subjected to Ehd1 specific quantitative real-time <t>PCR</t> (qRT-PCR). Expression level of Ehd1 in SV80 was defined as 100%. (B) Protein lysates prepared from CEDNIK (F165T, F110T) and control (SV80) fibroblasts were subjected to Western blot analysis and interacted with anti-EHD1 and anti-ERK antibodies, as a loading control. (C) Fibroblasts (SV80 and F110T) were transfected with GFP-EHD1, fixed and visualized by confocal microscopy. (D) RNA was isolated from SV80 and CEDNIK fibroblasts and subjected to Ehd2, Ehd3 or Ehd4 specific RT-PCR as detailed under Materials and Methods . (E) Control (SV80) and CEDNIK (F110T) fibroblasts were transfected with YFP-EHD2, GFP-EHD3 and YFP-EHD4 expressing plasmids for 18 h, after which they were fixed and visualized by confocal microscopy. No change in EHD1 RNA level, protein level or localization was observed in CEDNIK cells, compared to control cells. Also, no change in the intracellular localization or RNA levels of other EHDs was observed in CEDNIK cells. M- 1 kb marker. Bar,10 µm.
    Ez 10 Rna Isolation Kit, supplied by Bio Basic Canada, used in various techniques. Bioz Stars score: 89/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GENEFLOW LIMITED ez rna total rna isolation kit
    Expression of EHDs in CEDNIK fibroblasts. (A) <t>RNA</t> was isolated from control (SV80) and CEDNIK (F110T and F165T) fibroblasts and was subjected to Ehd1 specific quantitative real-time <t>PCR</t> (qRT-PCR). Expression level of Ehd1 in SV80 was defined as 100%. (B) Protein lysates prepared from CEDNIK (F165T, F110T) and control (SV80) fibroblasts were subjected to Western blot analysis and interacted with anti-EHD1 and anti-ERK antibodies, as a loading control. (C) Fibroblasts (SV80 and F110T) were transfected with GFP-EHD1, fixed and visualized by confocal microscopy. (D) RNA was isolated from SV80 and CEDNIK fibroblasts and subjected to Ehd2, Ehd3 or Ehd4 specific RT-PCR as detailed under Materials and Methods . (E) Control (SV80) and CEDNIK (F110T) fibroblasts were transfected with YFP-EHD2, GFP-EHD3 and YFP-EHD4 expressing plasmids for 18 h, after which they were fixed and visualized by confocal microscopy. No change in EHD1 RNA level, protein level or localization was observed in CEDNIK cells, compared to control cells. Also, no change in the intracellular localization or RNA levels of other EHDs was observed in CEDNIK cells. M- 1 kb marker. Bar,10 µm.
    Ez Rna Total Rna Isolation Kit, supplied by GENEFLOW LIMITED, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biological Industries Inc ez rna total rna dna protein isolation kit
    Expression of EHDs in CEDNIK fibroblasts. (A) <t>RNA</t> was isolated from control (SV80) and CEDNIK (F110T and F165T) fibroblasts and was subjected to Ehd1 specific quantitative real-time <t>PCR</t> (qRT-PCR). Expression level of Ehd1 in SV80 was defined as 100%. (B) Protein lysates prepared from CEDNIK (F165T, F110T) and control (SV80) fibroblasts were subjected to Western blot analysis and interacted with anti-EHD1 and anti-ERK antibodies, as a loading control. (C) Fibroblasts (SV80 and F110T) were transfected with GFP-EHD1, fixed and visualized by confocal microscopy. (D) RNA was isolated from SV80 and CEDNIK fibroblasts and subjected to Ehd2, Ehd3 or Ehd4 specific RT-PCR as detailed under Materials and Methods . (E) Control (SV80) and CEDNIK (F110T) fibroblasts were transfected with YFP-EHD2, GFP-EHD3 and YFP-EHD4 expressing plasmids for 18 h, after which they were fixed and visualized by confocal microscopy. No change in EHD1 RNA level, protein level or localization was observed in CEDNIK cells, compared to control cells. Also, no change in the intracellular localization or RNA levels of other EHDs was observed in CEDNIK cells. M- 1 kb marker. Bar,10 µm.
    Ez Rna Total Rna Dna Protein Isolation Kit, supplied by Biological Industries Inc, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sangon Biotech ez 10 spin column total rna isolation kit
    Expression of EHDs in CEDNIK fibroblasts. (A) <t>RNA</t> was isolated from control (SV80) and CEDNIK (F110T and F165T) fibroblasts and was subjected to Ehd1 specific quantitative real-time <t>PCR</t> (qRT-PCR). Expression level of Ehd1 in SV80 was defined as 100%. (B) Protein lysates prepared from CEDNIK (F165T, F110T) and control (SV80) fibroblasts were subjected to Western blot analysis and interacted with anti-EHD1 and anti-ERK antibodies, as a loading control. (C) Fibroblasts (SV80 and F110T) were transfected with GFP-EHD1, fixed and visualized by confocal microscopy. (D) RNA was isolated from SV80 and CEDNIK fibroblasts and subjected to Ehd2, Ehd3 or Ehd4 specific RT-PCR as detailed under Materials and Methods . (E) Control (SV80) and CEDNIK (F110T) fibroblasts were transfected with YFP-EHD2, GFP-EHD3 and YFP-EHD4 expressing plasmids for 18 h, after which they were fixed and visualized by confocal microscopy. No change in EHD1 RNA level, protein level or localization was observed in CEDNIK cells, compared to control cells. Also, no change in the intracellular localization or RNA levels of other EHDs was observed in CEDNIK cells. M- 1 kb marker. Bar,10 µm.
    Ez 10 Spin Column Total Rna Isolation Kit, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 90/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio Basic Canada ez 10 spin column total rna isolation kit
    Expression of EHDs in CEDNIK fibroblasts. (A) <t>RNA</t> was isolated from control (SV80) and CEDNIK (F110T and F165T) fibroblasts and was subjected to Ehd1 specific quantitative real-time <t>PCR</t> (qRT-PCR). Expression level of Ehd1 in SV80 was defined as 100%. (B) Protein lysates prepared from CEDNIK (F165T, F110T) and control (SV80) fibroblasts were subjected to Western blot analysis and interacted with anti-EHD1 and anti-ERK antibodies, as a loading control. (C) Fibroblasts (SV80 and F110T) were transfected with GFP-EHD1, fixed and visualized by confocal microscopy. (D) RNA was isolated from SV80 and CEDNIK fibroblasts and subjected to Ehd2, Ehd3 or Ehd4 specific RT-PCR as detailed under Materials and Methods . (E) Control (SV80) and CEDNIK (F110T) fibroblasts were transfected with YFP-EHD2, GFP-EHD3 and YFP-EHD4 expressing plasmids for 18 h, after which they were fixed and visualized by confocal microscopy. No change in EHD1 RNA level, protein level or localization was observed in CEDNIK cells, compared to control cells. Also, no change in the intracellular localization or RNA levels of other EHDs was observed in CEDNIK cells. M- 1 kb marker. Bar,10 µm.
    Ez 10 Spin Column Total Rna Isolation Kit, supplied by Bio Basic Canada, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biological Industries Inc ez rnaii isolation kit
    Expression of EHDs in CEDNIK fibroblasts. (A) <t>RNA</t> was isolated from control (SV80) and CEDNIK (F110T and F165T) fibroblasts and was subjected to Ehd1 specific quantitative real-time <t>PCR</t> (qRT-PCR). Expression level of Ehd1 in SV80 was defined as 100%. (B) Protein lysates prepared from CEDNIK (F165T, F110T) and control (SV80) fibroblasts were subjected to Western blot analysis and interacted with anti-EHD1 and anti-ERK antibodies, as a loading control. (C) Fibroblasts (SV80 and F110T) were transfected with GFP-EHD1, fixed and visualized by confocal microscopy. (D) RNA was isolated from SV80 and CEDNIK fibroblasts and subjected to Ehd2, Ehd3 or Ehd4 specific RT-PCR as detailed under Materials and Methods . (E) Control (SV80) and CEDNIK (F110T) fibroblasts were transfected with YFP-EHD2, GFP-EHD3 and YFP-EHD4 expressing plasmids for 18 h, after which they were fixed and visualized by confocal microscopy. No change in EHD1 RNA level, protein level or localization was observed in CEDNIK cells, compared to control cells. Also, no change in the intracellular localization or RNA levels of other EHDs was observed in CEDNIK cells. M- 1 kb marker. Bar,10 µm.
    Ez Rnaii Isolation Kit, supplied by Biological Industries Inc, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biological Industries Inc ez rna kit
    Induction of CCL2 and CLXL8 in TNFα-stimulated MSCs is not mediated via the AP-1 pathway. (A) Human BM-derived MSCs were stimulated by <t>TNF-α</t> (50 ng/ml) for 5 and 10 minutes. Control cells were treated by the vehicle of TNF-α. c-Jun levels and phosphorylation were determined by western blot (WB) analyses. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Human BM-derived MSCs were transiently transfected by small interfering <t>RNA</t> (siRNA) to c-Jun or by control siRNA. (B1) c-Jun expression was determined by WB analyses. β-Tubulin was used as loading control. (B2) Following siRNA transfection, the cells were stimulated by TNF-α (25 ng/ml; in this part of the study we used a suboptimal concentration of TNF-α in order to facilitate detection of inhibitory effects) for 24 hours. Expression levels of CCL2 and CXCL8 in the supernatants of the cells were determined by ELISA, in the linear range of absorbance. # siRNA to c-Jun has yielded minor increases or reductions in CCL2 and CXCL8 secretion in different experiments (see Results and discussion), and thus overall there was no significant effect on CCL2 and CXCL8 secretion. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results.
    Ez Rna Kit, supplied by Biological Industries Inc, used in various techniques. Bioz Stars score: 91/100, based on 338 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biological Industries Inc ez rna rna extraction kit
    Localization, stability and RNAi activity of intravitreally injected siCASP2. ( a ) Detection of Cy3 fluorescence in isolated RGC 1 h (red line) or 18 h (green line) following single intravitreal injection of 40 μ g Cy3-siRNA per rat eye. FACS analysis displaying results obtained from representative one out of two retinal tissue pools analysed per time point are shown. ( b ) Representative micro-autoradiographs of paraffin sections (counterstained with haematoxylin and eosin) from untreated ((i), (ii)) and siCASP2-injected rat eyes ((iii)–(iv)), which were hybridized with either siCASP2-specific ((i)–(iv)) or with a nonspecific ((v), (vi)) control 33 P-labelled probe ((i), (iii), (v)) – bright field images, ((ii), (iv), (vi)) – corresponding dark field images. In dark field images, hybridization signals are white dots over all retinal layers in sections of siCASP2-injected eyes hybridized to the specific probe ((iv)) and were especially prominent in the GCL (arrow). No signal in the GCL is detected in control sections ((ii), (vi)) (exposure 24 h, original magnification × 20). The eyes for in situ hybridization analysis were enucleated at 2 h after intravitreal injection of 20 μ g siRNA. At least 10 eyes from each group were analysed from 2 to 3 different experiments. ( c ) Quantification of siCASP2±S.D. in retina at different times after intravitreal injection of 20 μ g per eye siCASP2 ( n =6). Control retinas were obtained from intact non-injected eyes. ( d ) Detection of siCASP2-mediated RNAi using RLM-RACE: (i) EtBr-stained agarose gel with electrophoresed RLM-RACE products from siCASP2-transfected PC12 cells (size control) and from retinas collected at 4 h after intravitreal injection of <t>PBS</t> (1); 20 μ g siCNL (2); or 20 μ g siCASP2 (3) (yellow boxes indicate gel regions excised for cloning and subsequent colony sequencing); (ii) autoradiograph of the Southern blot of the gel shown in (i) after hybridization with radiolabelled RLM-RACE junction-specific probe; and (iii) example of the results of colony sequencing showing the RLM-RACE junction corresponding to the expected siCASP2 produced cleavage site. RLM-RACE was performed on <t>RNA</t> extracted from a pool of two retinas from eyes injected with PBS, pool of five retinas from the eyes injected with siCNL or a pool of four retinas from the eyes injected with siCASP2
    Ez Rna Rna Extraction Kit, supplied by Biological Industries Inc, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Qiagen ez 1 rna cell mini kit
    Localization, stability and RNAi activity of intravitreally injected siCASP2. ( a ) Detection of Cy3 fluorescence in isolated RGC 1 h (red line) or 18 h (green line) following single intravitreal injection of 40 μ g Cy3-siRNA per rat eye. FACS analysis displaying results obtained from representative one out of two retinal tissue pools analysed per time point are shown. ( b ) Representative micro-autoradiographs of paraffin sections (counterstained with haematoxylin and eosin) from untreated ((i), (ii)) and siCASP2-injected rat eyes ((iii)–(iv)), which were hybridized with either siCASP2-specific ((i)–(iv)) or with a nonspecific ((v), (vi)) control 33 P-labelled probe ((i), (iii), (v)) – bright field images, ((ii), (iv), (vi)) – corresponding dark field images. In dark field images, hybridization signals are white dots over all retinal layers in sections of siCASP2-injected eyes hybridized to the specific probe ((iv)) and were especially prominent in the GCL (arrow). No signal in the GCL is detected in control sections ((ii), (vi)) (exposure 24 h, original magnification × 20). The eyes for in situ hybridization analysis were enucleated at 2 h after intravitreal injection of 20 μ g siRNA. At least 10 eyes from each group were analysed from 2 to 3 different experiments. ( c ) Quantification of siCASP2±S.D. in retina at different times after intravitreal injection of 20 μ g per eye siCASP2 ( n =6). Control retinas were obtained from intact non-injected eyes. ( d ) Detection of siCASP2-mediated RNAi using RLM-RACE: (i) EtBr-stained agarose gel with electrophoresed RLM-RACE products from siCASP2-transfected PC12 cells (size control) and from retinas collected at 4 h after intravitreal injection of <t>PBS</t> (1); 20 μ g siCNL (2); or 20 μ g siCASP2 (3) (yellow boxes indicate gel regions excised for cloning and subsequent colony sequencing); (ii) autoradiograph of the Southern blot of the gel shown in (i) after hybridization with radiolabelled RLM-RACE junction-specific probe; and (iii) example of the results of colony sequencing showing the RLM-RACE junction corresponding to the expected siCASP2 produced cleavage site. RLM-RACE was performed on <t>RNA</t> extracted from a pool of two retinas from eyes injected with PBS, pool of five retinas from the eyes injected with siCNL or a pool of four retinas from the eyes injected with siCASP2
    Ez 1 Rna Cell Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 97/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher strip ez rna kit
    Localization, stability and RNAi activity of intravitreally injected siCASP2. ( a ) Detection of Cy3 fluorescence in isolated RGC 1 h (red line) or 18 h (green line) following single intravitreal injection of 40 μ g Cy3-siRNA per rat eye. FACS analysis displaying results obtained from representative one out of two retinal tissue pools analysed per time point are shown. ( b ) Representative micro-autoradiographs of paraffin sections (counterstained with haematoxylin and eosin) from untreated ((i), (ii)) and siCASP2-injected rat eyes ((iii)–(iv)), which were hybridized with either siCASP2-specific ((i)–(iv)) or with a nonspecific ((v), (vi)) control 33 P-labelled probe ((i), (iii), (v)) – bright field images, ((ii), (iv), (vi)) – corresponding dark field images. In dark field images, hybridization signals are white dots over all retinal layers in sections of siCASP2-injected eyes hybridized to the specific probe ((iv)) and were especially prominent in the GCL (arrow). No signal in the GCL is detected in control sections ((ii), (vi)) (exposure 24 h, original magnification × 20). The eyes for in situ hybridization analysis were enucleated at 2 h after intravitreal injection of 20 μ g siRNA. At least 10 eyes from each group were analysed from 2 to 3 different experiments. ( c ) Quantification of siCASP2±S.D. in retina at different times after intravitreal injection of 20 μ g per eye siCASP2 ( n =6). Control retinas were obtained from intact non-injected eyes. ( d ) Detection of siCASP2-mediated RNAi using RLM-RACE: (i) EtBr-stained agarose gel with electrophoresed RLM-RACE products from siCASP2-transfected PC12 cells (size control) and from retinas collected at 4 h after intravitreal injection of <t>PBS</t> (1); 20 μ g siCNL (2); or 20 μ g siCASP2 (3) (yellow boxes indicate gel regions excised for cloning and subsequent colony sequencing); (ii) autoradiograph of the Southern blot of the gel shown in (i) after hybridization with radiolabelled RLM-RACE junction-specific probe; and (iii) example of the results of colony sequencing showing the RLM-RACE junction corresponding to the expected siCASP2 produced cleavage site. RLM-RACE was performed on <t>RNA</t> extracted from a pool of two retinas from eyes injected with PBS, pool of five retinas from the eyes injected with siCNL or a pool of four retinas from the eyes injected with siCASP2
    Strip Ez Rna Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression of EHDs in CEDNIK fibroblasts. (A) RNA was isolated from control (SV80) and CEDNIK (F110T and F165T) fibroblasts and was subjected to Ehd1 specific quantitative real-time PCR (qRT-PCR). Expression level of Ehd1 in SV80 was defined as 100%. (B) Protein lysates prepared from CEDNIK (F165T, F110T) and control (SV80) fibroblasts were subjected to Western blot analysis and interacted with anti-EHD1 and anti-ERK antibodies, as a loading control. (C) Fibroblasts (SV80 and F110T) were transfected with GFP-EHD1, fixed and visualized by confocal microscopy. (D) RNA was isolated from SV80 and CEDNIK fibroblasts and subjected to Ehd2, Ehd3 or Ehd4 specific RT-PCR as detailed under Materials and Methods . (E) Control (SV80) and CEDNIK (F110T) fibroblasts were transfected with YFP-EHD2, GFP-EHD3 and YFP-EHD4 expressing plasmids for 18 h, after which they were fixed and visualized by confocal microscopy. No change in EHD1 RNA level, protein level or localization was observed in CEDNIK cells, compared to control cells. Also, no change in the intracellular localization or RNA levels of other EHDs was observed in CEDNIK cells. M- 1 kb marker. Bar,10 µm.

    Journal: PLoS ONE

    Article Title: Loss of SNAP29 Impairs Endocytic Recycling and Cell Motility

    doi: 10.1371/journal.pone.0009759

    Figure Lengend Snippet: Expression of EHDs in CEDNIK fibroblasts. (A) RNA was isolated from control (SV80) and CEDNIK (F110T and F165T) fibroblasts and was subjected to Ehd1 specific quantitative real-time PCR (qRT-PCR). Expression level of Ehd1 in SV80 was defined as 100%. (B) Protein lysates prepared from CEDNIK (F165T, F110T) and control (SV80) fibroblasts were subjected to Western blot analysis and interacted with anti-EHD1 and anti-ERK antibodies, as a loading control. (C) Fibroblasts (SV80 and F110T) were transfected with GFP-EHD1, fixed and visualized by confocal microscopy. (D) RNA was isolated from SV80 and CEDNIK fibroblasts and subjected to Ehd2, Ehd3 or Ehd4 specific RT-PCR as detailed under Materials and Methods . (E) Control (SV80) and CEDNIK (F110T) fibroblasts were transfected with YFP-EHD2, GFP-EHD3 and YFP-EHD4 expressing plasmids for 18 h, after which they were fixed and visualized by confocal microscopy. No change in EHD1 RNA level, protein level or localization was observed in CEDNIK cells, compared to control cells. Also, no change in the intracellular localization or RNA levels of other EHDs was observed in CEDNIK cells. M- 1 kb marker. Bar,10 µm.

    Article Snippet: Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) Total RNA was extracted from fibroblasts using the EZ-RNA isolation kit (Biological Industries, Beit Haemek, Israel).

    Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction, Marker

    Microarray results validation. T24 cells were infected with 50 MOI of either RAdnCD40L (AdnL) or RAdMock (AdM) or treated with sCD40L (sL) at a concentration of 1 µg/ ml for 24 hours or left untreated as a control (CNT). ( A ) Expression levels of the indicated transcripts were examined utilising cDNA synthesised from total RNA isolated from cells by qRT-PCR technique. Results represent the mean of three replicate pairs ± SD. ( B ) 50 µg of total cell lysates were examined by western blot analysis for IRF1, RASSF5 and CD40L expression, β-actin was examined to ensure equal inputs. ( C ) Correlation analysis of qPCR and microarray results of the qPCR-selected transcripts. ( D ) RT-PCR analysis of PTPRU, TOP2A, SLIT2, PADI2 and TRAF1 transcripts were also examined utilising cDNA as a template. GAPDH was examined to ensure equal cDNA input across different treatment.

    Journal: Scientific Reports

    Article Title: CD40L membrane retention enhances the immunostimulatory effects of CD40 ligation

    doi: 10.1038/s41598-019-57293-y

    Figure Lengend Snippet: Microarray results validation. T24 cells were infected with 50 MOI of either RAdnCD40L (AdnL) or RAdMock (AdM) or treated with sCD40L (sL) at a concentration of 1 µg/ ml for 24 hours or left untreated as a control (CNT). ( A ) Expression levels of the indicated transcripts were examined utilising cDNA synthesised from total RNA isolated from cells by qRT-PCR technique. Results represent the mean of three replicate pairs ± SD. ( B ) 50 µg of total cell lysates were examined by western blot analysis for IRF1, RASSF5 and CD40L expression, β-actin was examined to ensure equal inputs. ( C ) Correlation analysis of qPCR and microarray results of the qPCR-selected transcripts. ( D ) RT-PCR analysis of PTPRU, TOP2A, SLIT2, PADI2 and TRAF1 transcripts were also examined utilising cDNA as a template. GAPDH was examined to ensure equal cDNA input across different treatment.

    Article Snippet: RT-PCR analysis Using the RETROscript RNase reverse transcription kit (Ambion Europe, Huntingdon, UK) according to the manufacturer’s instructions, cDNA was synthesised from total RNA extracted by EZ-RNA total isolation kit (Biological Industries, Israel).

    Techniques: Microarray, Infection, Concentration Assay, Expressing, Isolation, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    Specific expression of GAP 10 in premolt gastrolith disc as demonstrated by RT-PCR ( A ) and localization of GAP 10 expression to the columnar epithelium forming the gastrolith by in situ hybridization ( B ). Total RNA was extracted from the gastrolith disc,

    Journal: The Journal of Biological Chemistry

    Article Title: A Protein Involved in the Assembly of an Extracellular Calcium Storage Matrix *

    doi: 10.1074/jbc.M109.071068

    Figure Lengend Snippet: Specific expression of GAP 10 in premolt gastrolith disc as demonstrated by RT-PCR ( A ) and localization of GAP 10 expression to the columnar epithelium forming the gastrolith by in situ hybridization ( B ). Total RNA was extracted from the gastrolith disc,

    Article Snippet: Total RNAs of the gastrolith disc and muscle tissue were extracted from premolt-induced males using EZ-RNA total RNA isolation kit (Biological Industries, Beit Haemek, Israel). cDNA was prepared from 1 μg of total RNA using the Super SMART PCR cDNA synthesis kit (Clontech).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization

    Proliferation, migration and survival inhibition of MiaPaCa2 cells following treatment with APA nanocarrier containing miRNA–siRNA combination. a miR-34a levels in MiaPaCa2 cells following treatment with APA polyplexes containing miR-34a or NC-miR, quantified relative to U6 RNA using qRT-PCR, showing in vitro delivery efficacy by APA nanocarrier. Untreated cells served as control. b miR-34a’s target genes (CDK6, MET, Notch, and Bcl2) levels 48 h following the same treatment as in a . c PLK1 mRNA levels following treatment with APA polyplexes containing PLK1-siRNA or NC-siRNA for 24 h, quantified relative to GAPDH mRNA using qRT-PCR. d PLK1 protein levels following the same treatment as in c . Densitometric analysis of western blot is presented as percentage of band intensity compared to untreated cells (unit). e – g Proliferation of MiaPaCa2 cells following treatment with APA polyplexes containing different concentrations of miR-34a or NC-miR e , PLK1-siRNA or NC-siRNA f , or miR-34a (100 nM) and PLK1-siRNA (50 nM) combination g . Statistical significance is shown for the comparison between miR-34a and NC-miR in e and between PLK1-siRNA and NC-siRNA in f ( n = 3). h , i Migration of MiaPaCa2 cells 48 h following incubation with the same treatments as in g . Representative images of the cells from 0 to 48 h time points h quantified as wound confluence (percent out of initial wound at time 0) using the IncuCyte software i . (2 biological repeats were done in triplicates). j Representative images of cell survival via colony formation assay for 11 days. k Quantification of colonies from 3 biological repeats as their total area relative to untreated cells (control) using ImageJ software (experiments were done in triplicates). Data represent mean ± SD. (Student’s t -test, NS; not significant for P > 0.05, * P

    Journal: Nature Communications

    Article Title: Amphiphilic nanocarrier-induced modulation of PLK1 and miR-34a leads to improved therapeutic response in pancreatic cancer

    doi: 10.1038/s41467-017-02283-9

    Figure Lengend Snippet: Proliferation, migration and survival inhibition of MiaPaCa2 cells following treatment with APA nanocarrier containing miRNA–siRNA combination. a miR-34a levels in MiaPaCa2 cells following treatment with APA polyplexes containing miR-34a or NC-miR, quantified relative to U6 RNA using qRT-PCR, showing in vitro delivery efficacy by APA nanocarrier. Untreated cells served as control. b miR-34a’s target genes (CDK6, MET, Notch, and Bcl2) levels 48 h following the same treatment as in a . c PLK1 mRNA levels following treatment with APA polyplexes containing PLK1-siRNA or NC-siRNA for 24 h, quantified relative to GAPDH mRNA using qRT-PCR. d PLK1 protein levels following the same treatment as in c . Densitometric analysis of western blot is presented as percentage of band intensity compared to untreated cells (unit). e – g Proliferation of MiaPaCa2 cells following treatment with APA polyplexes containing different concentrations of miR-34a or NC-miR e , PLK1-siRNA or NC-siRNA f , or miR-34a (100 nM) and PLK1-siRNA (50 nM) combination g . Statistical significance is shown for the comparison between miR-34a and NC-miR in e and between PLK1-siRNA and NC-siRNA in f ( n = 3). h , i Migration of MiaPaCa2 cells 48 h following incubation with the same treatments as in g . Representative images of the cells from 0 to 48 h time points h quantified as wound confluence (percent out of initial wound at time 0) using the IncuCyte software i . (2 biological repeats were done in triplicates). j Representative images of cell survival via colony formation assay for 11 days. k Quantification of colonies from 3 biological repeats as their total area relative to untreated cells (control) using ImageJ software (experiments were done in triplicates). Data represent mean ± SD. (Student’s t -test, NS; not significant for P > 0.05, * P

    Article Snippet: miRNA/mRNA expression levels determination Total RNA from cultured cells was isolated using EZ-RNA II total RNA isolation kit (Biological Industries) according to the manufacturer’s instructions.

    Techniques: Migration, Inhibition, Quantitative RT-PCR, In Vitro, Western Blot, Incubation, Software, Colony Assay

    Induction of CCL2 and CLXL8 in TNFα-stimulated MSCs is not mediated via the AP-1 pathway. (A) Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 5 and 10 minutes. Control cells were treated by the vehicle of TNF-α. c-Jun levels and phosphorylation were determined by western blot (WB) analyses. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Human BM-derived MSCs were transiently transfected by small interfering RNA (siRNA) to c-Jun or by control siRNA. (B1) c-Jun expression was determined by WB analyses. β-Tubulin was used as loading control. (B2) Following siRNA transfection, the cells were stimulated by TNF-α (25 ng/ml; in this part of the study we used a suboptimal concentration of TNF-α in order to facilitate detection of inhibitory effects) for 24 hours. Expression levels of CCL2 and CXCL8 in the supernatants of the cells were determined by ELISA, in the linear range of absorbance. # siRNA to c-Jun has yielded minor increases or reductions in CCL2 and CXCL8 secretion in different experiments (see Results and discussion), and thus overall there was no significant effect on CCL2 and CXCL8 secretion. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results.

    Journal: Stem Cell Research & Therapy

    Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

    doi: 10.1186/s13287-015-0080-7

    Figure Lengend Snippet: Induction of CCL2 and CLXL8 in TNFα-stimulated MSCs is not mediated via the AP-1 pathway. (A) Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 5 and 10 minutes. Control cells were treated by the vehicle of TNF-α. c-Jun levels and phosphorylation were determined by western blot (WB) analyses. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Human BM-derived MSCs were transiently transfected by small interfering RNA (siRNA) to c-Jun or by control siRNA. (B1) c-Jun expression was determined by WB analyses. β-Tubulin was used as loading control. (B2) Following siRNA transfection, the cells were stimulated by TNF-α (25 ng/ml; in this part of the study we used a suboptimal concentration of TNF-α in order to facilitate detection of inhibitory effects) for 24 hours. Expression levels of CCL2 and CXCL8 in the supernatants of the cells were determined by ELISA, in the linear range of absorbance. # siRNA to c-Jun has yielded minor increases or reductions in CCL2 and CXCL8 secretion in different experiments (see Results and discussion), and thus overall there was no significant effect on CCL2 and CXCL8 secretion. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results.

    Article Snippet: Polymerase chain reactions (PCR) To determine the expression of human TNF receptors, total RNA was isolated by the EZ-RNA kit (#20-400-100; Biological Industries) from patient CAFs #1 and CAFs #2 isolates, from MSCs and from HL-60 cells that served as positive control.

    Techniques: Derivative Assay, Western Blot, Transfection, Small Interfering RNA, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Localization, stability and RNAi activity of intravitreally injected siCASP2. ( a ) Detection of Cy3 fluorescence in isolated RGC 1 h (red line) or 18 h (green line) following single intravitreal injection of 40 μ g Cy3-siRNA per rat eye. FACS analysis displaying results obtained from representative one out of two retinal tissue pools analysed per time point are shown. ( b ) Representative micro-autoradiographs of paraffin sections (counterstained with haematoxylin and eosin) from untreated ((i), (ii)) and siCASP2-injected rat eyes ((iii)–(iv)), which were hybridized with either siCASP2-specific ((i)–(iv)) or with a nonspecific ((v), (vi)) control 33 P-labelled probe ((i), (iii), (v)) – bright field images, ((ii), (iv), (vi)) – corresponding dark field images. In dark field images, hybridization signals are white dots over all retinal layers in sections of siCASP2-injected eyes hybridized to the specific probe ((iv)) and were especially prominent in the GCL (arrow). No signal in the GCL is detected in control sections ((ii), (vi)) (exposure 24 h, original magnification × 20). The eyes for in situ hybridization analysis were enucleated at 2 h after intravitreal injection of 20 μ g siRNA. At least 10 eyes from each group were analysed from 2 to 3 different experiments. ( c ) Quantification of siCASP2±S.D. in retina at different times after intravitreal injection of 20 μ g per eye siCASP2 ( n =6). Control retinas were obtained from intact non-injected eyes. ( d ) Detection of siCASP2-mediated RNAi using RLM-RACE: (i) EtBr-stained agarose gel with electrophoresed RLM-RACE products from siCASP2-transfected PC12 cells (size control) and from retinas collected at 4 h after intravitreal injection of PBS (1); 20 μ g siCNL (2); or 20 μ g siCASP2 (3) (yellow boxes indicate gel regions excised for cloning and subsequent colony sequencing); (ii) autoradiograph of the Southern blot of the gel shown in (i) after hybridization with radiolabelled RLM-RACE junction-specific probe; and (iii) example of the results of colony sequencing showing the RLM-RACE junction corresponding to the expected siCASP2 produced cleavage site. RLM-RACE was performed on RNA extracted from a pool of two retinas from eyes injected with PBS, pool of five retinas from the eyes injected with siCNL or a pool of four retinas from the eyes injected with siCASP2

    Journal: Cell Death & Disease

    Article Title: Ocular neuroprotection by siRNA targeting caspase-2

    doi: 10.1038/cddis.2011.54

    Figure Lengend Snippet: Localization, stability and RNAi activity of intravitreally injected siCASP2. ( a ) Detection of Cy3 fluorescence in isolated RGC 1 h (red line) or 18 h (green line) following single intravitreal injection of 40 μ g Cy3-siRNA per rat eye. FACS analysis displaying results obtained from representative one out of two retinal tissue pools analysed per time point are shown. ( b ) Representative micro-autoradiographs of paraffin sections (counterstained with haematoxylin and eosin) from untreated ((i), (ii)) and siCASP2-injected rat eyes ((iii)–(iv)), which were hybridized with either siCASP2-specific ((i)–(iv)) or with a nonspecific ((v), (vi)) control 33 P-labelled probe ((i), (iii), (v)) – bright field images, ((ii), (iv), (vi)) – corresponding dark field images. In dark field images, hybridization signals are white dots over all retinal layers in sections of siCASP2-injected eyes hybridized to the specific probe ((iv)) and were especially prominent in the GCL (arrow). No signal in the GCL is detected in control sections ((ii), (vi)) (exposure 24 h, original magnification × 20). The eyes for in situ hybridization analysis were enucleated at 2 h after intravitreal injection of 20 μ g siRNA. At least 10 eyes from each group were analysed from 2 to 3 different experiments. ( c ) Quantification of siCASP2±S.D. in retina at different times after intravitreal injection of 20 μ g per eye siCASP2 ( n =6). Control retinas were obtained from intact non-injected eyes. ( d ) Detection of siCASP2-mediated RNAi using RLM-RACE: (i) EtBr-stained agarose gel with electrophoresed RLM-RACE products from siCASP2-transfected PC12 cells (size control) and from retinas collected at 4 h after intravitreal injection of PBS (1); 20 μ g siCNL (2); or 20 μ g siCASP2 (3) (yellow boxes indicate gel regions excised for cloning and subsequent colony sequencing); (ii) autoradiograph of the Southern blot of the gel shown in (i) after hybridization with radiolabelled RLM-RACE junction-specific probe; and (iii) example of the results of colony sequencing showing the RLM-RACE junction corresponding to the expected siCASP2 produced cleavage site. RLM-RACE was performed on RNA extracted from a pool of two retinas from eyes injected with PBS, pool of five retinas from the eyes injected with siCNL or a pool of four retinas from the eyes injected with siCASP2

    Article Snippet: Whole retinas were washed in a large volume of ice-cold PBS and RNA was extracted using EZ-RNA RNA extraction kit (Biological Industries, Kibbutz Beit-Haemek, Israel).

    Techniques: Activity Assay, Injection, Fluorescence, Isolation, FACS, Hybridization, In Situ Hybridization, Staining, Agarose Gel Electrophoresis, Transfection, Clone Assay, Sequencing, Autoradiography, Southern Blot, Produced