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    Qiagen rnase free dnase set
    Extracellular vesicle internalization and transfer of GFP mRNA ( A ) Super resolution 3D reconstruction of GFP+ mRPC following 24 h incubation with PKH26 labeled extracellular vesicles. Red vesicles are visibly localized near the cell surface and within cytoplasm. In the XZ axis, GFP (green), EVs (red) and nuclei (blue, DAPI). ( B ) same as ( A ) with GFP (FITC) channel removed to increase visibility of PKH26 (TRITC) labeled EVs. Each panel contains three cross-sectional views (xy, xz, and yz). Scale: 5 µm. ( C ) RT-PCR analysis of GFP mRNA transfer between GFP+ mRPCs and non-GFP hRPCs. Non-GFP hRPCs served as negative control; GFP+ mRPCs served as postive control. GAPDH served as the internal control gene. EVs were treated using an <t>RNase-Free</t> <t>DNase</t> Set to remove DNA comtamination before cDNA synthesis. ( D ) Intensities of RT-PCR images were measured with ImageJ software and normalized to GAPDH. Relative levels of hRPC GFP after transfer of EVs is significantly higher than negative control.
    Rnase Free Dnase Set, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Extracellular vesicle internalization and transfer of GFP mRNA ( A ) Super resolution 3D reconstruction of GFP+ mRPC following 24 h incubation with PKH26 labeled extracellular vesicles. Red vesicles are visibly localized near the cell surface and within cytoplasm. In the XZ axis, GFP (green), EVs (red) and nuclei (blue, DAPI). ( B ) same as ( A ) with GFP (FITC) channel removed to increase visibility of PKH26 (TRITC) labeled EVs. Each panel contains three cross-sectional views (xy, xz, and yz). Scale: 5 µm. ( C ) RT-PCR analysis of GFP mRNA transfer between GFP+ mRPCs and non-GFP hRPCs. Non-GFP hRPCs served as negative control; GFP+ mRPCs served as postive control. GAPDH served as the internal control gene. EVs were treated using an RNase-Free DNase Set to remove DNA comtamination before cDNA synthesis. ( D ) Intensities of RT-PCR images were measured with ImageJ software and normalized to GAPDH. Relative levels of hRPC GFP after transfer of EVs is significantly higher than negative control.

    Journal: Scientific Reports

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins

    doi: 10.1038/s41598-018-20421-1

    Figure Lengend Snippet: Extracellular vesicle internalization and transfer of GFP mRNA ( A ) Super resolution 3D reconstruction of GFP+ mRPC following 24 h incubation with PKH26 labeled extracellular vesicles. Red vesicles are visibly localized near the cell surface and within cytoplasm. In the XZ axis, GFP (green), EVs (red) and nuclei (blue, DAPI). ( B ) same as ( A ) with GFP (FITC) channel removed to increase visibility of PKH26 (TRITC) labeled EVs. Each panel contains three cross-sectional views (xy, xz, and yz). Scale: 5 µm. ( C ) RT-PCR analysis of GFP mRNA transfer between GFP+ mRPCs and non-GFP hRPCs. Non-GFP hRPCs served as negative control; GFP+ mRPCs served as postive control. GAPDH served as the internal control gene. EVs were treated using an RNase-Free DNase Set to remove DNA comtamination before cDNA synthesis. ( D ) Intensities of RT-PCR images were measured with ImageJ software and normalized to GAPDH. Relative levels of hRPC GFP after transfer of EVs is significantly higher than negative control.

    Article Snippet: DNA contamination was removed using an RNase-Free DNase Set (Qiagen). cDNA was synthesized using equal amounts of RNA samples (800 ng), according to the AMV First Strand cDNA Synthesis Kit instructions (NEB Biolabs). β-actin and GAPDH were used as a housekeeping gene controls for mRNA analysis , .

    Techniques: Incubation, Labeling, Reverse Transcription Polymerase Chain Reaction, Negative Control, Software

    Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Journal: Frontiers in Microbiology

    Article Title: Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    doi: 10.3389/fmicb.2016.01588

    Figure Lengend Snippet: Suggested DNA/RNA co-extraction workflow for environmental samples, with stronger emphasis on thorough purification prior to all enzymatic steps (including DNase digestion). Optional steps are indicated by dotted arrows. Note that RNase digestion (between Extracts II and III) may be necessary for better results downstream, but may be omitted as a separate step (in the current study, RNase is present in the qPCR mix). (A) Pre-lysis inhibitor removal is only advisable if quick methods are used, or if mRNA is not the target molecule (lengthy inhibitor removal procedures compromise RNA integrity). (B) Various methods may be used, such as phenol/chloroform procedures or nucleic acid precipitation. (C) This purification step should target the removal of enzymatic-inhibitors (e.g., humic/fulvic acids and polyphenolics). (D) Purification of partially digested RNA extracts with residual genomic DNA aids in the removal of enduring inhibitors, prior to further digestion. (E) Stringent and well-documented quality control via rigorous and sensitive detection (preferably quantitative methods) is necessary to detect residual amplifiable gDNA prior to reverse transcription.

    Article Snippet: The following DNases were tested for their ability to remove amplifiable DNA from TNA samples: DNase I (Sigma), RNase-Free DNase Set (QIAGEN), RNase-Free DNase I (Epicentre Biotechnologies) and TURBO DNA-free DNase Kit (Ambion, Life Technologies).

    Techniques: Environmental Sampling, Purification, Real-time Polymerase Chain Reaction, Lysis

    Presence of stable miRNAs in Drosophila melanogaster hemolymph. ( A , B ) Clear HL droplets extruded from fly head and thorax. ( C – F ) Real-time qPCR amplification of selected HL miRNAs and mRNAs. Total RNA including small RNA was extracted from HL samples and analyzed with qPCR to measure the levels of miRNAs and mRNAs. The y -axis represents the relative fluorescence units (RFU) in a semi-log scale. The x -axis represents the cycle at which fluorescence was detected above an automatically determined threshold. ( C ) Amplification plots for miR-14, miR-8, and miR-184 measured in a representative HL sample. ( D ) Amplification plots for miR-14, let-7, bantam, and spiked-in synthetic C. elegans cel-miR-39 RNA in representative HL sample. ( E ) The amplification plots for tubulin, actin, and gapdh mRNAs determined by qPCR using total RNA from HL and S2 cells. Sample from S2 cells are used as a positive control for detecting Drosophila mRNAs by qPCR. The amplification curves of all three mRNAs are superimposed on one another, reflecting the presence of similar amounts of these mRNA in S2 cells. Amplification curves from HL samples show that fluorescent products appear after about 30 cycles, reflecting the significantly lower abundance of these mRNAs in HL relative to S2 cells. ( F ) The cycle threshold (Ct) fold-change of selected miRNA amplified in the absence or presence of RNase A and DNase I. Total RNA was extracted from HL samples spiked with 10 fmoles of cel-miR-39 RNA. The x -axis represents the ratio of raw Ct values from control samples divided by raw Ct values from samples incubated with RNase A and DNase I. The significantly higher magnitude of the Ct fold change of the spiked-in synthetic miRNA relative to those of the miRNA indicates that the HL-miRNA are present in nuclease-resistant, stable form.

    Journal: Genomics Insights

    Article Title: MicroRNAs Circulate in the Hemolymph of Drosophila and Accumulate Relative to Tissue microRNAs in an Age-Dependent Manner

    doi: 10.4137/GEI.S38147

    Figure Lengend Snippet: Presence of stable miRNAs in Drosophila melanogaster hemolymph. ( A , B ) Clear HL droplets extruded from fly head and thorax. ( C – F ) Real-time qPCR amplification of selected HL miRNAs and mRNAs. Total RNA including small RNA was extracted from HL samples and analyzed with qPCR to measure the levels of miRNAs and mRNAs. The y -axis represents the relative fluorescence units (RFU) in a semi-log scale. The x -axis represents the cycle at which fluorescence was detected above an automatically determined threshold. ( C ) Amplification plots for miR-14, miR-8, and miR-184 measured in a representative HL sample. ( D ) Amplification plots for miR-14, let-7, bantam, and spiked-in synthetic C. elegans cel-miR-39 RNA in representative HL sample. ( E ) The amplification plots for tubulin, actin, and gapdh mRNAs determined by qPCR using total RNA from HL and S2 cells. Sample from S2 cells are used as a positive control for detecting Drosophila mRNAs by qPCR. The amplification curves of all three mRNAs are superimposed on one another, reflecting the presence of similar amounts of these mRNA in S2 cells. Amplification curves from HL samples show that fluorescent products appear after about 30 cycles, reflecting the significantly lower abundance of these mRNAs in HL relative to S2 cells. ( F ) The cycle threshold (Ct) fold-change of selected miRNA amplified in the absence or presence of RNase A and DNase I. Total RNA was extracted from HL samples spiked with 10 fmoles of cel-miR-39 RNA. The x -axis represents the ratio of raw Ct values from control samples divided by raw Ct values from samples incubated with RNase A and DNase I. The significantly higher magnitude of the Ct fold change of the spiked-in synthetic miRNA relative to those of the miRNA indicates that the HL-miRNA are present in nuclease-resistant, stable form.

    Article Snippet: HL samples, collected and prepared as described above, were spiked with 10 fmoles of synthetic Caenorhabditis elegans cel-miR-39 miScript miRNA (Qiagen #MSY0000010) and incubated for 15 minutes at 37°C with and without addition of 100 ng/mL of RNase A (Thermo Scientific #EN0531) and 4 units of DNase I (Qiagen, #79254).

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Fluorescence, Positive Control, Incubation