rneasy micro kit  (Qiagen)

 
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    Name:
    RNeasy Micro Kit
    Description:
    For purification of up to 45 µg total RNA from cell and tissue samples Kit contents Qiagen RNeasy Micro Kit 50 preps 10 to 14L Elution Volume 5mg Sample Tissue Cells Sample Total RNA Purification Spin Column Format Silica Technology Ideal for Northern Dot and Slot Blotting End point RT PCR Quantitative Real time RT PCR Includes 50 RNeasy MinElute Spin Columns Collection Tubes 1 5mL and 2mL RNase free DNase I Carrier RNA RNase free Reagents and Buffers Benefits Fast procedure delivering high quality total RNA in minutes Ready to use RNA for high performance in any downstream application Consistent RNA yields from very small amounts of starting material No phenol chloroform extraction No CsCl gradients no LiCl or ethanol precipitation
    Catalog Number:
    74004
    Price:
    492
    Category:
    RNeasy Micro Kit
    Buy from Supplier


    Structured Review

    Qiagen rneasy micro kit
    RNeasy Micro Kit
    For purification of up to 45 µg total RNA from cell and tissue samples Kit contents Qiagen RNeasy Micro Kit 50 preps 10 to 14L Elution Volume 5mg Sample Tissue Cells Sample Total RNA Purification Spin Column Format Silica Technology Ideal for Northern Dot and Slot Blotting End point RT PCR Quantitative Real time RT PCR Includes 50 RNeasy MinElute Spin Columns Collection Tubes 1 5mL and 2mL RNase free DNase I Carrier RNA RNase free Reagents and Buffers Benefits Fast procedure delivering high quality total RNA in minutes Ready to use RNA for high performance in any downstream application Consistent RNA yields from very small amounts of starting material No phenol chloroform extraction No CsCl gradients no LiCl or ethanol precipitation
    https://www.bioz.com/result/rneasy micro kit/product/Qiagen
    Average 99 stars, based on 1653 article reviews
    Price from $9.99 to $1999.99
    rneasy micro kit - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells"

    Article Title: Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034778

    Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.
    Figure Legend Snippet: Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.

    Techniques Used: Isolation, Polymerase Chain Reaction, Expressing, TRAP Assay, Activity Assay

    2) Product Images from "Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells"

    Article Title: Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034778

    Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.
    Figure Legend Snippet: Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.

    Techniques Used: Isolation, Polymerase Chain Reaction, Expressing, TRAP Assay, Activity Assay

    3) Product Images from "Ionic currents in intimal cultured synoviocytes from the rabbit"

    Article Title: Ionic currents in intimal cultured synoviocytes from the rabbit

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00028.2010

    Cells from passage 6 (the passage used for electrophysiological studies) were subjected to total RNA extraction using the RNeasy Micro Kit. Total RNA was also prepared from freshly microdissected synovium using the TRIzol method. A : the transcription product was amplified with primers specific for hyaluronan synthase 2 (HAS2), and the resulting DNA bands are shown. HAS2 message was evident in both the passage 6 -cultured synoviocytes (Cult Syn P6) and in intact synovium at dilutions of 1:1 and 1:5 but absent from the nontemplate control (NTC). B, bottom : fixed erythrocyte exclusion test. Under normal conditions the synoviocytes were surrounded by a clear area from which erythrocytes were excluded. This clear area disappeared after hyaluronidase addition, suggesting that it was due to hyaluronan secretion by the synoviocyte. Rab, rabbit; P4H, prolyl 4-hydroxylase. Black calibration bar represents 20 μm in each case.
    Figure Legend Snippet: Cells from passage 6 (the passage used for electrophysiological studies) were subjected to total RNA extraction using the RNeasy Micro Kit. Total RNA was also prepared from freshly microdissected synovium using the TRIzol method. A : the transcription product was amplified with primers specific for hyaluronan synthase 2 (HAS2), and the resulting DNA bands are shown. HAS2 message was evident in both the passage 6 -cultured synoviocytes (Cult Syn P6) and in intact synovium at dilutions of 1:1 and 1:5 but absent from the nontemplate control (NTC). B, bottom : fixed erythrocyte exclusion test. Under normal conditions the synoviocytes were surrounded by a clear area from which erythrocytes were excluded. This clear area disappeared after hyaluronidase addition, suggesting that it was due to hyaluronan secretion by the synoviocyte. Rab, rabbit; P4H, prolyl 4-hydroxylase. Black calibration bar represents 20 μm in each case.

    Techniques Used: RNA Extraction, Amplification, Cell Culture

    4) Product Images from "Gene Expression Analysis of In Vivo Fluorescent Cells"

    Article Title: Gene Expression Analysis of In Vivo Fluorescent Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001151

    RNA analysis by the Bioanalyzer 2100. ( A ) RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method described here. ( B ) RNA isolated from frozen tissue by the optimized proteinase K/acid phenol method. ( C ) RNA isolated from fixed tissue by TRIzol method. ( D ) RNA isolated from fixed tissue by RNeasy Micro Kit. (E) One round of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( F ) One round of amplification of Ambion Control RNA. ( G ) Two rounds of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( H ) RNA ladder: first peak is RNA marker, next mark 200, 500, 1000, 2000, 4000 and 6000 nt.
    Figure Legend Snippet: RNA analysis by the Bioanalyzer 2100. ( A ) RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method described here. ( B ) RNA isolated from frozen tissue by the optimized proteinase K/acid phenol method. ( C ) RNA isolated from fixed tissue by TRIzol method. ( D ) RNA isolated from fixed tissue by RNeasy Micro Kit. (E) One round of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( F ) One round of amplification of Ambion Control RNA. ( G ) Two rounds of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( H ) RNA ladder: first peak is RNA marker, next mark 200, 500, 1000, 2000, 4000 and 6000 nt.

    Techniques Used: Isolation, Amplification, Marker

    5) Product Images from "Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate"

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201606382

    Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.
    Figure Legend Snippet: Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Serial Dilution, Incubation, Purification

    6) Product Images from "Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate"

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201606382

    Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.
    Figure Legend Snippet: Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Serial Dilution, Incubation, Purification

    7) Product Images from "Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease"

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

    Journal: Kidney international

    doi: 10.1038/ki.2010.106

    Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.
    Figure Legend Snippet: Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.

    Techniques Used: Lysis, Isolation, RNA Extraction, Incubation

    8) Product Images from "Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate"

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201606382

    Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.
    Figure Legend Snippet: Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Serial Dilution, Incubation, Purification

    9) Product Images from "High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos"

    Article Title: High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0172171

    Bioanalyzer Agilent electrophoresis runs. Examples of representative Bioanalyzer Agilent electrophoresis runs for the five different methods applied for RNA extractions from P . lividus embryos: TRIzol, GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) and Aurum™ Total RNA Mini Kit (Biorad). Four different numerical amounts of embryos were used for RNA extraction: 500, 1000, 2500 and 5000 embryos. The ladder (L) is reported in the first lane of each run. The green band at the bottom of each panel is the RNA 6000 Nano Marker (Agilent RNA 6000 Nano Kit, Agilent Technologies, Inc.). Red box in the 500 lane of TRIzol indicates that the Bioanalyzer software cannot calculate RIN values (reported as N/A in the Table 1 ) for this sample, because of very low concentration and high level of degradation of the RNA.
    Figure Legend Snippet: Bioanalyzer Agilent electrophoresis runs. Examples of representative Bioanalyzer Agilent electrophoresis runs for the five different methods applied for RNA extractions from P . lividus embryos: TRIzol, GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) and Aurum™ Total RNA Mini Kit (Biorad). Four different numerical amounts of embryos were used for RNA extraction: 500, 1000, 2500 and 5000 embryos. The ladder (L) is reported in the first lane of each run. The green band at the bottom of each panel is the RNA 6000 Nano Marker (Agilent RNA 6000 Nano Kit, Agilent Technologies, Inc.). Red box in the 500 lane of TRIzol indicates that the Bioanalyzer software cannot calculate RIN values (reported as N/A in the Table 1 ) for this sample, because of very low concentration and high level of degradation of the RNA.

    Techniques Used: Electrophoresis, RNA Extraction, Marker, Software, Concentration Assay

    Agilent Bioanlyzer electropherograms. Examples of representative Agilent Bioanlyzer electropherograms of P . lividus RNA: for TRIzol, GenElute and RNAqueous RNA extraction from 5000 embryos extraction; for RNeasy and Aurum RNA extraction from 2500 embryos (see also Table 1 ). Relative Fluorescent Unit (FU) and seconds of migration (s) of RNA samples isolated according to the five different extraction methods are reported. RIN values are also reported.
    Figure Legend Snippet: Agilent Bioanlyzer electropherograms. Examples of representative Agilent Bioanlyzer electropherograms of P . lividus RNA: for TRIzol, GenElute and RNAqueous RNA extraction from 5000 embryos extraction; for RNeasy and Aurum RNA extraction from 2500 embryos (see also Table 1 ). Relative Fluorescent Unit (FU) and seconds of migration (s) of RNA samples isolated according to the five different extraction methods are reported. RIN values are also reported.

    Techniques Used: RNA Extraction, Migration, Isolation

    10) Product Images from "Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease"

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

    Journal: Kidney international

    doi: 10.1038/ki.2010.106

    RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.
    Figure Legend Snippet: RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.

    Techniques Used: Isolation

    Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.
    Figure Legend Snippet: Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.

    Techniques Used: Lysis, Isolation, RNA Extraction, Incubation

    11) Product Images from "Overexpression of Colligin 2 in Glioma Vasculature is Associated with Overexpression of Heat Shock Factor 2"

    Article Title: Overexpression of Colligin 2 in Glioma Vasculature is Associated with Overexpression of Heat Shock Factor 2

    Journal: Gene Regulation and Systems Biology

    doi: 10.4137/GRSB.S4546

    mRNA expression of colligin 2, HSF1, 2 and 3, collagen 1, CD31 and NG2 in low- and high-grade glioma and normal control brain. Data in this figure are the average ± SD of one representative experiment with 4 tissues in each group. Expression data are presented relative to the average mRNA expression levels measured in total RNA isolated from normal brain tissues (n = 4). Prior to isolation, all tissues were assessed by a qualified pathologist to ensure the origin and quality of the tissues. Total RNA was isolated with the RNeasy Micro kit (Qiagen BV, Venlo, The Netherlands). cDNA was prepared by use of the RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, St Leon-Rot, Germany). The resulting cDNA preparations were analyzed by real-time PCR with TaqMan gene expression assays and TaqMan Universal PCR Master Mix (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). PCRs were performed in a 20 μL reaction volume in an Applied BioSystems 7900HT Fast Real-Time PCR system. Negative controls included minus RT and H 2 O-only samples, which showed to be negative in all cases. The most stable mRNA set for our 4 tissue groups were calculated with NormFinder 19 with the Datan Framework GenEx Pro package version 4.3.2 and was shown to be a combination of GUSB, HMBS, HPRT1 and NOXA1 . Expression of GUSB , HMBS , HPRT1 and NOXA1 was therefore used as a reference to control sample loading and RNA quality, as described previously. 20 Differences in mRNA concentrations were determined by the non parametric Kruskal-Wallis test with P
    Figure Legend Snippet: mRNA expression of colligin 2, HSF1, 2 and 3, collagen 1, CD31 and NG2 in low- and high-grade glioma and normal control brain. Data in this figure are the average ± SD of one representative experiment with 4 tissues in each group. Expression data are presented relative to the average mRNA expression levels measured in total RNA isolated from normal brain tissues (n = 4). Prior to isolation, all tissues were assessed by a qualified pathologist to ensure the origin and quality of the tissues. Total RNA was isolated with the RNeasy Micro kit (Qiagen BV, Venlo, The Netherlands). cDNA was prepared by use of the RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, St Leon-Rot, Germany). The resulting cDNA preparations were analyzed by real-time PCR with TaqMan gene expression assays and TaqMan Universal PCR Master Mix (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). PCRs were performed in a 20 μL reaction volume in an Applied BioSystems 7900HT Fast Real-Time PCR system. Negative controls included minus RT and H 2 O-only samples, which showed to be negative in all cases. The most stable mRNA set for our 4 tissue groups were calculated with NormFinder 19 with the Datan Framework GenEx Pro package version 4.3.2 and was shown to be a combination of GUSB, HMBS, HPRT1 and NOXA1 . Expression of GUSB , HMBS , HPRT1 and NOXA1 was therefore used as a reference to control sample loading and RNA quality, as described previously. 20 Differences in mRNA concentrations were determined by the non parametric Kruskal-Wallis test with P

    Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    12) Product Images from "Gene Expression Analysis of In Vivo Fluorescent Cells"

    Article Title: Gene Expression Analysis of In Vivo Fluorescent Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001151

    RNA analysis by the Bioanalyzer 2100. ( A ) RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method described here. ( B ) RNA isolated from frozen tissue by the optimized proteinase K/acid phenol method. ( C ) RNA isolated from fixed tissue by TRIzol method. ( D ) RNA isolated from fixed tissue by RNeasy Micro Kit. (E) One round of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( F ) One round of amplification of Ambion Control RNA. ( G ) Two rounds of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( H ) RNA ladder: first peak is RNA marker, next mark 200, 500, 1000, 2000, 4000 and 6000 nt.
    Figure Legend Snippet: RNA analysis by the Bioanalyzer 2100. ( A ) RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method described here. ( B ) RNA isolated from frozen tissue by the optimized proteinase K/acid phenol method. ( C ) RNA isolated from fixed tissue by TRIzol method. ( D ) RNA isolated from fixed tissue by RNeasy Micro Kit. (E) One round of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( F ) One round of amplification of Ambion Control RNA. ( G ) Two rounds of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( H ) RNA ladder: first peak is RNA marker, next mark 200, 500, 1000, 2000, 4000 and 6000 nt.

    Techniques Used: Isolation, Amplification, Marker

    13) Product Images from "High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos"

    Article Title: High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0172171

    Bioanalyzer Agilent electrophoresis runs. Examples of representative Bioanalyzer Agilent electrophoresis runs for the five different methods applied for RNA extractions from P . lividus embryos: TRIzol, GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) and Aurum™ Total RNA Mini Kit (Biorad). Four different numerical amounts of embryos were used for RNA extraction: 500, 1000, 2500 and 5000 embryos. The ladder (L) is reported in the first lane of each run. The green band at the bottom of each panel is the RNA 6000 Nano Marker (Agilent RNA 6000 Nano Kit, Agilent Technologies, Inc.). Red box in the 500 lane of TRIzol indicates that the Bioanalyzer software cannot calculate RIN values (reported as N/A in the Table 1 ) for this sample, because of very low concentration and high level of degradation of the RNA.
    Figure Legend Snippet: Bioanalyzer Agilent electrophoresis runs. Examples of representative Bioanalyzer Agilent electrophoresis runs for the five different methods applied for RNA extractions from P . lividus embryos: TRIzol, GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) and Aurum™ Total RNA Mini Kit (Biorad). Four different numerical amounts of embryos were used for RNA extraction: 500, 1000, 2500 and 5000 embryos. The ladder (L) is reported in the first lane of each run. The green band at the bottom of each panel is the RNA 6000 Nano Marker (Agilent RNA 6000 Nano Kit, Agilent Technologies, Inc.). Red box in the 500 lane of TRIzol indicates that the Bioanalyzer software cannot calculate RIN values (reported as N/A in the Table 1 ) for this sample, because of very low concentration and high level of degradation of the RNA.

    Techniques Used: Electrophoresis, RNA Extraction, Marker, Software, Concentration Assay

    Agilent Bioanlyzer electropherograms. Examples of representative Agilent Bioanlyzer electropherograms of P . lividus RNA: for TRIzol, GenElute and RNAqueous RNA extraction from 5000 embryos extraction; for RNeasy and Aurum RNA extraction from 2500 embryos (see also Table 1 ). Relative Fluorescent Unit (FU) and seconds of migration (s) of RNA samples isolated according to the five different extraction methods are reported. RIN values are also reported.
    Figure Legend Snippet: Agilent Bioanlyzer electropherograms. Examples of representative Agilent Bioanlyzer electropherograms of P . lividus RNA: for TRIzol, GenElute and RNAqueous RNA extraction from 5000 embryos extraction; for RNeasy and Aurum RNA extraction from 2500 embryos (see also Table 1 ). Relative Fluorescent Unit (FU) and seconds of migration (s) of RNA samples isolated according to the five different extraction methods are reported. RIN values are also reported.

    Techniques Used: RNA Extraction, Migration, Isolation

    14) Product Images from "Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in Rheumatoid Arthritis"

    Article Title: Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in Rheumatoid Arthritis

    Journal: bioRxiv

    doi: 10.1101/2020.03.08.970046

    Reduction of α2-6 sialylation and ST6Gal1 levels is observed only in sublining CD90+ synovial fibroblasts. a) Synovial fibroblasts from healthy mice were isolated from mouse joints and identified by flow cytometry as in Fig. 1 (Zombie Violet-, CD45-, CD31-, Podoplanin+), separated into CD90+ and CD90-populations and stained with the biotinylated lectins SNA, MAAII and PNA and streptevadin-PE-Cy7. Graphs show the Mean Fluorescence Intensity for each cytokine and each dot represent cells from one individual mouse (n=3). b) Synovial fibroblasts from healthy and arthritic mice (n=4) were isolated from mouse joints as in (a) and RNA was purified using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. Expression of IL-6, MMP3 and ST6Gal1 mRNA was quantified by RT-qPCR. Statistics: one tail unpaired t-test was used for statistics, **p
    Figure Legend Snippet: Reduction of α2-6 sialylation and ST6Gal1 levels is observed only in sublining CD90+ synovial fibroblasts. a) Synovial fibroblasts from healthy mice were isolated from mouse joints and identified by flow cytometry as in Fig. 1 (Zombie Violet-, CD45-, CD31-, Podoplanin+), separated into CD90+ and CD90-populations and stained with the biotinylated lectins SNA, MAAII and PNA and streptevadin-PE-Cy7. Graphs show the Mean Fluorescence Intensity for each cytokine and each dot represent cells from one individual mouse (n=3). b) Synovial fibroblasts from healthy and arthritic mice (n=4) were isolated from mouse joints as in (a) and RNA was purified using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. Expression of IL-6, MMP3 and ST6Gal1 mRNA was quantified by RT-qPCR. Statistics: one tail unpaired t-test was used for statistics, **p

    Techniques Used: Mouse Assay, Isolation, Flow Cytometry, Staining, Fluorescence, Purification, Expressing, Quantitative RT-PCR

    15) Product Images from "Direct Cell Lysis for Single-Cell Gene Expression Profiling"

    Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling

    Journal: Frontiers in Oncology

    doi: 10.3389/fonc.2013.00274

    Comparison of direct cell lysis and column based extraction . (A) One to 2048 cells in steps of two were FACS-sorted and mRNA was extracted either by direct cell lysis or RNeasy Micro columns for RT-qPCR analysis. The difference in Cq-values (left y -axis) between 1 mg/ml BSA and column based extraction reflects the difference in sensitivity between the two methods. Percentage of samples with detectable cDNA is plotted on the right y -axis. The dynamic range for the endogenously expressed genes is shown by linear curve fits. The “Spike only” control sample shows the effect of column based extraction of the spikes alone without any cell material. Data are shown as mean ± SD ( n = 4) (see also Table S7 in Supplementary Material). (B) Comparison of direct cell lysis to column based extraction for all transcripts. The mean difference in sensitivity is shown by averaging the expression of Gapdh , Vim , Dll , and Jag1 . The dotted line, at a value of one, indicates when column based extraction is as efficient as direct lysis. Missing data were replaced with a Cq-value of 40.
    Figure Legend Snippet: Comparison of direct cell lysis and column based extraction . (A) One to 2048 cells in steps of two were FACS-sorted and mRNA was extracted either by direct cell lysis or RNeasy Micro columns for RT-qPCR analysis. The difference in Cq-values (left y -axis) between 1 mg/ml BSA and column based extraction reflects the difference in sensitivity between the two methods. Percentage of samples with detectable cDNA is plotted on the right y -axis. The dynamic range for the endogenously expressed genes is shown by linear curve fits. The “Spike only” control sample shows the effect of column based extraction of the spikes alone without any cell material. Data are shown as mean ± SD ( n = 4) (see also Table S7 in Supplementary Material). (B) Comparison of direct cell lysis to column based extraction for all transcripts. The mean difference in sensitivity is shown by averaging the expression of Gapdh , Vim , Dll , and Jag1 . The dotted line, at a value of one, indicates when column based extraction is as efficient as direct lysis. Missing data were replaced with a Cq-value of 40.

    Techniques Used: Lysis, FACS, Quantitative RT-PCR, Expressing

    16) Product Images from "Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease"

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

    Journal: Kidney international

    doi: 10.1038/ki.2010.106

    RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.
    Figure Legend Snippet: RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.

    Techniques Used: Isolation

    Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.
    Figure Legend Snippet: Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.

    Techniques Used: Lysis, Isolation, RNA Extraction, Incubation

    17) Product Images from "Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq"

    Article Title: Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00185

    Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p
    Figure Legend Snippet: Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p

    Techniques Used: Laser Capture Microdissection, Isolation

    18) Product Images from "Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq"

    Article Title: Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00185

    Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p
    Figure Legend Snippet: Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p

    Techniques Used: Laser Capture Microdissection, Isolation

    19) Product Images from "Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq"

    Article Title: Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00185

    Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p
    Figure Legend Snippet: Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p

    Techniques Used: Laser Capture Microdissection, Isolation

    20) Product Images from "Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease"

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

    Journal: Kidney international

    doi: 10.1038/ki.2010.106

    RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.
    Figure Legend Snippet: RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.

    Techniques Used: Isolation

    Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.
    Figure Legend Snippet: Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.

    Techniques Used: Lysis, Isolation, RNA Extraction, Incubation

    21) Product Images from "Ionic currents in intimal cultured synoviocytes from the rabbit"

    Article Title: Ionic currents in intimal cultured synoviocytes from the rabbit

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00028.2010

    Cells from passage 6 (the passage used for electrophysiological studies) were subjected to total RNA extraction using the RNeasy Micro Kit. Total RNA was also prepared from freshly microdissected synovium using the TRIzol method. A : the transcription product was amplified with primers specific for hyaluronan synthase 2 (HAS2), and the resulting DNA bands are shown. HAS2 message was evident in both the passage 6 -cultured synoviocytes (Cult Syn P6) and in intact synovium at dilutions of 1:1 and 1:5 but absent from the nontemplate control (NTC). B, bottom : fixed erythrocyte exclusion test. Under normal conditions the synoviocytes were surrounded by a clear area from which erythrocytes were excluded. This clear area disappeared after hyaluronidase addition, suggesting that it was due to hyaluronan secretion by the synoviocyte. Rab, rabbit; P4H, prolyl 4-hydroxylase. Black calibration bar represents 20 μm in each case.
    Figure Legend Snippet: Cells from passage 6 (the passage used for electrophysiological studies) were subjected to total RNA extraction using the RNeasy Micro Kit. Total RNA was also prepared from freshly microdissected synovium using the TRIzol method. A : the transcription product was amplified with primers specific for hyaluronan synthase 2 (HAS2), and the resulting DNA bands are shown. HAS2 message was evident in both the passage 6 -cultured synoviocytes (Cult Syn P6) and in intact synovium at dilutions of 1:1 and 1:5 but absent from the nontemplate control (NTC). B, bottom : fixed erythrocyte exclusion test. Under normal conditions the synoviocytes were surrounded by a clear area from which erythrocytes were excluded. This clear area disappeared after hyaluronidase addition, suggesting that it was due to hyaluronan secretion by the synoviocyte. Rab, rabbit; P4H, prolyl 4-hydroxylase. Black calibration bar represents 20 μm in each case.

    Techniques Used: RNA Extraction, Amplification, Cell Culture

    22) Product Images from "A novel procedure for the quantitative analysis of metabolites, storage products and transcripts of laser microdissected seed tissues of Brassica napus"

    Article Title: A novel procedure for the quantitative analysis of metabolites, storage products and transcripts of laser microdissected seed tissues of Brassica napus

    Journal: Plant Methods

    doi: 10.1186/1746-4811-7-19

    Transcript analysis . Closeness of expression signals obtained after microarray hybridisation of conventionally extracted total RNA and amplified RNA samples of conventional, column-based and bead-based extraction methods. ( a c ) Electropherograms reflecting quality of total RNA, isolated with RNeasy ® (a) and mRNA, isolated with Dynabeads ® (c) from a 20 μm seed cryosection (43. DAF). ( b d ) Electropherograms of mRNA from the same samples (a/c) after 2 rounds of amplification. ( e ) Portions of signal intensities obtained from each hybridisation and divided into six groups. Y-axis represent % of observation. ( f ) Scatterplots comparing log 2 -transformed means of expression signals from the different sample treatments. Means are filtered against low signal intensities (≤ 10) and high coefficients of variation (≥ 20%).
    Figure Legend Snippet: Transcript analysis . Closeness of expression signals obtained after microarray hybridisation of conventionally extracted total RNA and amplified RNA samples of conventional, column-based and bead-based extraction methods. ( a c ) Electropherograms reflecting quality of total RNA, isolated with RNeasy ® (a) and mRNA, isolated with Dynabeads ® (c) from a 20 μm seed cryosection (43. DAF). ( b d ) Electropherograms of mRNA from the same samples (a/c) after 2 rounds of amplification. ( e ) Portions of signal intensities obtained from each hybridisation and divided into six groups. Y-axis represent % of observation. ( f ) Scatterplots comparing log 2 -transformed means of expression signals from the different sample treatments. Means are filtered against low signal intensities (≤ 10) and high coefficients of variation (≥ 20%).

    Techniques Used: Expressing, Microarray, Hybridization, Amplification, Isolation, Transformation Assay

    23) Product Images from "An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells"

    Article Title: An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00402

    RNA extraction evaluation. (A) Bioanalyser analysis of RNA Integritry Number (RIN), and nanospectrophotometer analysis of yield and concentration were obtained using three commercially-available ex traction kits with (+) or without post-extraction RNA purification and concentration. (B) RT-qPCR analysis of IFN- γ, RPL13a, SDHA , and TBP expression normalized to cell number (copies/10 6 cells) or cDNA concentration (copies/μL). 1 × 10 6 PBMCs paired samples were cultured with complete media (white) or PMA/Iono (gray) for 6 h. RNA was extracted using the RNeasy® Mini (Mini) Kit, RNeasy® Micro (Micro) Kit (both QIAGEN), or MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems), with concentration step performed using the RNeasy® MiniElute Cleanup Kit (QIAGEN). All samples were reverse transcribed with Superscript™ III (Invitrogen). Data were analyzed using a two-way ANOVA with post-hoc Bonferroni's multiple-comparisons test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Biological replicate ( n = 4), triplicate RNA extractions, with single reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the technical RNA extractions which were in turn calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.
    Figure Legend Snippet: RNA extraction evaluation. (A) Bioanalyser analysis of RNA Integritry Number (RIN), and nanospectrophotometer analysis of yield and concentration were obtained using three commercially-available ex traction kits with (+) or without post-extraction RNA purification and concentration. (B) RT-qPCR analysis of IFN- γ, RPL13a, SDHA , and TBP expression normalized to cell number (copies/10 6 cells) or cDNA concentration (copies/μL). 1 × 10 6 PBMCs paired samples were cultured with complete media (white) or PMA/Iono (gray) for 6 h. RNA was extracted using the RNeasy® Mini (Mini) Kit, RNeasy® Micro (Micro) Kit (both QIAGEN), or MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems), with concentration step performed using the RNeasy® MiniElute Cleanup Kit (QIAGEN). All samples were reverse transcribed with Superscript™ III (Invitrogen). Data were analyzed using a two-way ANOVA with post-hoc Bonferroni's multiple-comparisons test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Biological replicate ( n = 4), triplicate RNA extractions, with single reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the technical RNA extractions which were in turn calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.

    Techniques Used: RNA Extraction, Concentration Assay, Purification, Quantitative RT-PCR, Expressing, Cell Culture, Isolation, Real-time Polymerase Chain Reaction

    Reverse transcription evaluation. Four reverse transcription kits were evalued for relative qPCR signal for (A) maximal RNA yield, or (B) maximal RNA concentration. When maximizing RNA yield, RNA was extracted with MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems); when maximizing concentration, RNA was concentrated with RNeasy® MiniElute Cleanup Kit (QIAGEN). RNA was reverse transcribed with either Superscript™ III (SSIII), Superscript™ IV (SSIV) (both Invitrogen), iScript™ Advanced (iScript) (BioRad) or High-Capacity (HC) (ThermoFisher) reverse transcription kits. RNA was extracted from 1 × 10 6 PBMCs pared samples, cultured with complete media (white) or PMA/Iono (gray) for 6 h, then IFN- γ, RPL13a, SDHA , and TBP mRNA expression was quantified. Data were compared with a two-way ANOVA (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001 for post-hoc Bonferroni's multiple-comparisons test). Biological replicate ( n = 4), single RNA extractions, with triplicate reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the reverse transcription reactions calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.
    Figure Legend Snippet: Reverse transcription evaluation. Four reverse transcription kits were evalued for relative qPCR signal for (A) maximal RNA yield, or (B) maximal RNA concentration. When maximizing RNA yield, RNA was extracted with MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems); when maximizing concentration, RNA was concentrated with RNeasy® MiniElute Cleanup Kit (QIAGEN). RNA was reverse transcribed with either Superscript™ III (SSIII), Superscript™ IV (SSIV) (both Invitrogen), iScript™ Advanced (iScript) (BioRad) or High-Capacity (HC) (ThermoFisher) reverse transcription kits. RNA was extracted from 1 × 10 6 PBMCs pared samples, cultured with complete media (white) or PMA/Iono (gray) for 6 h, then IFN- γ, RPL13a, SDHA , and TBP mRNA expression was quantified. Data were compared with a two-way ANOVA (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001 for post-hoc Bonferroni's multiple-comparisons test). Biological replicate ( n = 4), single RNA extractions, with triplicate reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the reverse transcription reactions calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.

    Techniques Used: Real-time Polymerase Chain Reaction, Concentration Assay, Isolation, Cell Culture, Expressing

    Assay analytical sensitivity. Relative RT-qPCR signal for IFN- γ, RPL13a, SDHA , and TBP mRNA expression from log 10 dilutions of unstimulated PBMCs when (A) maximizing RNA yield, or (B) maximizing RNA concentration. When maximizing RNA yield, RNA was extracted with MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems); when maximizing concentration, RNA was concentrated with RNeasy® MiniElute Cleanup Kit (QIAGEN). All samples were reverse transcribed with Superscript™ IV (Invitrogen). mRNA expression was determined by absolute-quantitative RT-qPCR and gene copy number per reaction was normalized to log 10 copies per reaction. Biological replicate ( n = 3), single RNA extractions, with single reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.
    Figure Legend Snippet: Assay analytical sensitivity. Relative RT-qPCR signal for IFN- γ, RPL13a, SDHA , and TBP mRNA expression from log 10 dilutions of unstimulated PBMCs when (A) maximizing RNA yield, or (B) maximizing RNA concentration. When maximizing RNA yield, RNA was extracted with MagMAX™ mirVana ™ (MagMAX) Total RNA Isolation Kit (Applied Biosystems); when maximizing concentration, RNA was concentrated with RNeasy® MiniElute Cleanup Kit (QIAGEN). All samples were reverse transcribed with Superscript™ IV (Invitrogen). mRNA expression was determined by absolute-quantitative RT-qPCR and gene copy number per reaction was normalized to log 10 copies per reaction. Biological replicate ( n = 3), single RNA extractions, with single reverse transcription reactions per extraction were performed. Sample mean calculated from the mean of the technical triplicate qPCR reactions. Biological mean ± biological SEM are shown.

    Techniques Used: Quantitative RT-PCR, Expressing, Concentration Assay, Isolation, Real-time Polymerase Chain Reaction

    qPCR optimization. (A) Experimental workflow for qPCR optimization. (B) Effect of stimulation on mRNA expression of reference genes RPL13a, SDHA , and TBP . 1 × 10 6 PBMCs paired samples were cultured with complete media (white), or stimulated with PMA/Iono control (gray) for 0, 6, 12, 16, 24, or 48 h. RNA was extracted using the RNeasy® Mini (Mini) Kit, and reverse transcribed with Superscript™ III (Invitrogen). RNA expression was determined by absolute quantitative RT-qPCR wherein number of gene copies per reaction was quantified by standard curve and normalized to cell number. Data were compared with a two-way ANOVA with post-hoc Bonferroni's multiple-comparisons test (** P ≤ 0.01; *** P ≤ 0.001). Biological replicate ( n = 3) single RNA extractions with single reverse transcription reactions per extraction were performed. Sample mean calculated from technical triplicate qPCR. Biological mean ± biological SEM are shown.
    Figure Legend Snippet: qPCR optimization. (A) Experimental workflow for qPCR optimization. (B) Effect of stimulation on mRNA expression of reference genes RPL13a, SDHA , and TBP . 1 × 10 6 PBMCs paired samples were cultured with complete media (white), or stimulated with PMA/Iono control (gray) for 0, 6, 12, 16, 24, or 48 h. RNA was extracted using the RNeasy® Mini (Mini) Kit, and reverse transcribed with Superscript™ III (Invitrogen). RNA expression was determined by absolute quantitative RT-qPCR wherein number of gene copies per reaction was quantified by standard curve and normalized to cell number. Data were compared with a two-way ANOVA with post-hoc Bonferroni's multiple-comparisons test (** P ≤ 0.01; *** P ≤ 0.001). Biological replicate ( n = 3) single RNA extractions with single reverse transcription reactions per extraction were performed. Sample mean calculated from technical triplicate qPCR. Biological mean ± biological SEM are shown.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Cell Culture, RNA Expression, Quantitative RT-PCR

    24) Product Images from "Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes"

    Article Title: Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017625

    Experimental workflow to assess efficiency of NuGen probe generation technologies using low amounts of input RNA. HUVEC total RNA was titrated to cover a range of input RNA from 50 ng–10 pg. 50 ng (n = 1), 500 pg (n = 2) and 250 pg (n = 2) of total RNA was used as input for the WT-Ovation FFPE system V2 while 500 pg (n = 2), 250 pg (n = 2), 100 pg (n = 2), 50 pg (n = 2) and 10 pg (n = 2) were used as input for the WT-Ovation One-Direct system (NuGen Technologies, Inc). All cDNA reactions were purified via Zymo Research Clean and Concentrator™-25 or Qiagen RNeasy MinElute Cleanup kits (WT-Ovation FFPE V2 and WT-Ovation One-Direct systems respectively) as recommended. All purified cDNA probes were assessed for quantity and quality using the Agilent 2100 Bioanalyzer and the Nanodrop-8000 RNA Nano chips. FL-Ovation™ cDNA Biotin Module V2 (NuGEN) was used for fragmentation and biotin labelling of 5 µg of cDNA and used for subsequent hybridisation to Affymetrix HGU133 Plus 2.0 microarrays.
    Figure Legend Snippet: Experimental workflow to assess efficiency of NuGen probe generation technologies using low amounts of input RNA. HUVEC total RNA was titrated to cover a range of input RNA from 50 ng–10 pg. 50 ng (n = 1), 500 pg (n = 2) and 250 pg (n = 2) of total RNA was used as input for the WT-Ovation FFPE system V2 while 500 pg (n = 2), 250 pg (n = 2), 100 pg (n = 2), 50 pg (n = 2) and 10 pg (n = 2) were used as input for the WT-Ovation One-Direct system (NuGen Technologies, Inc). All cDNA reactions were purified via Zymo Research Clean and Concentrator™-25 or Qiagen RNeasy MinElute Cleanup kits (WT-Ovation FFPE V2 and WT-Ovation One-Direct systems respectively) as recommended. All purified cDNA probes were assessed for quantity and quality using the Agilent 2100 Bioanalyzer and the Nanodrop-8000 RNA Nano chips. FL-Ovation™ cDNA Biotin Module V2 (NuGEN) was used for fragmentation and biotin labelling of 5 µg of cDNA and used for subsequent hybridisation to Affymetrix HGU133 Plus 2.0 microarrays.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Purification, Hybridization

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    Article Snippet: .. Ten days post-differentiation, EBs in the supernatant were harvested by centrifugation (BeckmanAllegra-6R, 1000 rpm, 5 min) and RNA was isolated using the RNeasy Micro Kit (Qiagen). .. Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT)12–18 and used as template in subsequent PCR with Taq DNA Polymerase.

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    Article Snippet: .. Ten days post-differentiation, EBs in the supernatant were harvested by centrifugation (BeckmanAllegra-6R, 1000 rpm, 5 min) and RNA was isolated using the RNeasy Micro Kit (Qiagen). .. Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT)12–18 and used as template in subsequent PCR with Taq DNA Polymerase.

    Article Title: Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes
    Article Snippet: .. Total RNA from minimal cell numbers was also extracted using the RNeasy Micro RNA isolation kit with the following modifications (process B) ( ). ..

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    Article Title: Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes
    Article Snippet: .. RNA isolation from vascular endothelial biopsies was also carried out using the RNeasy Micro RNA isolation kit (Qiagen) following process B ( ). ..

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    Purification:

    Article Title: Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq
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    Real-time Polymerase Chain Reaction:

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    Qiagen rneasy kit
    2′- O methylation of internal adenosine. (A) Incorporation of 3 H-methyl into polyA. Homopolymer RNAs (1 µg) were incubated with 2 µg of DENV-4 MTase in the presence of [ 3 H-methyl]-SAM. After the methylation reaction, the un-incorporated [ 3 H-methyl]-SAM was removed by <t>RNeasy</t> kit. The amount of 3 H-methyl incorporation was measured by a MicroBeta counting. (B) SPA-based methylation analysis of oligo (A) 12 , (Am) 12 , and (m 6 ,m 6 A) 12 . All three <t>RNA</t> oligos were 3′-end biotinylated to facilitate SPA analysis. Am indicates that the 2′-OH of adenosine is methylated. m 6 ,m 6 A indicates that the amino N 6 position of adenosine is double methylated. (C) SPA-based methylation analysis of DENV-1 RNA. pppA-RNAs, representing the 5′ 211 nt of DENV-1 genome, were in vitro transcribed using biotinylated-CTP plus unmodified ATP, 2′- O -methyladenosine triphosphate (AmTP), or N 6 methyl adenosine triphosphate (m 6 ATP). The transcription reactions generated pppA-RNA, ppp(Am)-RNA, and ppp(m 6 A)RNA, respectively. The RNAs were then subjected to SPA-based internal methylation analysis. Average results and standard deviations from three independent experiments are presented.
    Rneasy Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 4510 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen mirna easy mini kit
    Alterations in cardiac <t>microRNA</t> expression 16–26 days after percutaneous cardiac delivery of scAAV6-U6-shRNA-PLB ( n =3) compared with control ( n =3). Microarray analysis revealed significant alterations in expression of two <t>miRNAs</t> after overexpression of shRNA. No significant changes were noted in several other miRNAs with previously defined roles in the heart. *Significant, as defined by p
    Mirna Easy Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    2′- O methylation of internal adenosine. (A) Incorporation of 3 H-methyl into polyA. Homopolymer RNAs (1 µg) were incubated with 2 µg of DENV-4 MTase in the presence of [ 3 H-methyl]-SAM. After the methylation reaction, the un-incorporated [ 3 H-methyl]-SAM was removed by RNeasy kit. The amount of 3 H-methyl incorporation was measured by a MicroBeta counting. (B) SPA-based methylation analysis of oligo (A) 12 , (Am) 12 , and (m 6 ,m 6 A) 12 . All three RNA oligos were 3′-end biotinylated to facilitate SPA analysis. Am indicates that the 2′-OH of adenosine is methylated. m 6 ,m 6 A indicates that the amino N 6 position of adenosine is double methylated. (C) SPA-based methylation analysis of DENV-1 RNA. pppA-RNAs, representing the 5′ 211 nt of DENV-1 genome, were in vitro transcribed using biotinylated-CTP plus unmodified ATP, 2′- O -methyladenosine triphosphate (AmTP), or N 6 methyl adenosine triphosphate (m 6 ATP). The transcription reactions generated pppA-RNA, ppp(Am)-RNA, and ppp(m 6 A)RNA, respectively. The RNAs were then subjected to SPA-based internal methylation analysis. Average results and standard deviations from three independent experiments are presented.

    Journal: PLoS Pathogens

    Article Title: 2?-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

    doi: 10.1371/journal.ppat.1002642

    Figure Lengend Snippet: 2′- O methylation of internal adenosine. (A) Incorporation of 3 H-methyl into polyA. Homopolymer RNAs (1 µg) were incubated with 2 µg of DENV-4 MTase in the presence of [ 3 H-methyl]-SAM. After the methylation reaction, the un-incorporated [ 3 H-methyl]-SAM was removed by RNeasy kit. The amount of 3 H-methyl incorporation was measured by a MicroBeta counting. (B) SPA-based methylation analysis of oligo (A) 12 , (Am) 12 , and (m 6 ,m 6 A) 12 . All three RNA oligos were 3′-end biotinylated to facilitate SPA analysis. Am indicates that the 2′-OH of adenosine is methylated. m 6 ,m 6 A indicates that the amino N 6 position of adenosine is double methylated. (C) SPA-based methylation analysis of DENV-1 RNA. pppA-RNAs, representing the 5′ 211 nt of DENV-1 genome, were in vitro transcribed using biotinylated-CTP plus unmodified ATP, 2′- O -methyladenosine triphosphate (AmTP), or N 6 methyl adenosine triphosphate (m 6 ATP). The transcription reactions generated pppA-RNA, ppp(Am)-RNA, and ppp(m 6 A)RNA, respectively. The RNAs were then subjected to SPA-based internal methylation analysis. Average results and standard deviations from three independent experiments are presented.

    Article Snippet: For each time point, total cellular RNA was extracted using RNeasy kit (Qiagen).

    Techniques: Methylation, Incubation, In Vitro, Generated

    Quantitative RT-PCR analysis of PAI-1 mRNA levels in patients. The total RNA was harvested from the peripheral blood mononuclear cells using an RNeasy kit according to the manufacturer’s instructions. The RT-PCR experiments were repeated at least 3 times. RNA was reverse transcribed into cDNA using random primers in a Reverse Transcription II system according to the manufacturer’s instructions. The expression of PAI-1 mRNA was quantified by quantitative PCR using an ABI Prism Sequence Detection System. Template-negative and RT-negative conditions were used as controls. Amplification of the endogenous GAPDH cDNA was monitored. The levels (mean value) of PAI-1 transcripts in patients were calculated. RT-PCR, reverse-transcription-PCR, PAI-1, plasminogen activator inhibitor-1.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Therapeutic effects of calcium dobesilate on diabetic nephropathy mediated through reduction of expression of PAI-1

    doi: 10.3892/etm.2012.755

    Figure Lengend Snippet: Quantitative RT-PCR analysis of PAI-1 mRNA levels in patients. The total RNA was harvested from the peripheral blood mononuclear cells using an RNeasy kit according to the manufacturer’s instructions. The RT-PCR experiments were repeated at least 3 times. RNA was reverse transcribed into cDNA using random primers in a Reverse Transcription II system according to the manufacturer’s instructions. The expression of PAI-1 mRNA was quantified by quantitative PCR using an ABI Prism Sequence Detection System. Template-negative and RT-negative conditions were used as controls. Amplification of the endogenous GAPDH cDNA was monitored. The levels (mean value) of PAI-1 transcripts in patients were calculated. RT-PCR, reverse-transcription-PCR, PAI-1, plasminogen activator inhibitor-1.

    Article Snippet: The total RNA was harvested from peripheral blood mononuclear cells using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

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

    SPP1 expression is up-regulated in glioma initiating cells ( A ) Representative examples of dot plots and histograms of Rhod 123(–) subpopulations from 5 glioma cell lines were acquired by flow cytometry. Glioma cells (1 × 10 7 ) were trypsinized and incubated with 0.1 μg/ml Rhod123 for 20 min in 37°C. After washing the cells were placed in 37°C for 90 min for Rhod123 exclusion in a dark compartment. Cells kept on ice to inhibit exclusion of Rhod123 were used as a positive control for gating in flow cytometry. Fractions of Rhod123(+) and Rhod123(–) cells were sorted using FACS Aria and a left panel shows a gating strategy. ( B ) Analysis of SPP1, OCT3/4 and NANOG gene expression in Rhod 123(–) subpopulations sorted from four human glioma cell lines. Total RNA was isolated from sorted cells using Qiagen RNeasy kit and the levels of SPP1, NANOG and OCT3/4 mRNA were determined by qPCR in Rhod 123(+) and Rhod 123(–) subpopulations; their expression in Rhod 123(+) subpopulations was taken as 1 (a red line). P values were calculated with a t-test and considered significant when *P ≤ 0.05 and * *P ≤ 0.01. ( C ) Quantification of the expression of Spp1, Oct3/4 and Nanog in Rhod 123(–) subpopulations isolated from rat C6 glioma cells versus their levels in Rhod 123(+) subpopulations taken as 1 (a red line); n = 3.

    Journal: Oncotarget

    Article Title: The embryonic type of SPP1 transcriptional regulation is re-activated in glioblastoma

    doi: 10.18632/oncotarget.14092

    Figure Lengend Snippet: SPP1 expression is up-regulated in glioma initiating cells ( A ) Representative examples of dot plots and histograms of Rhod 123(–) subpopulations from 5 glioma cell lines were acquired by flow cytometry. Glioma cells (1 × 10 7 ) were trypsinized and incubated with 0.1 μg/ml Rhod123 for 20 min in 37°C. After washing the cells were placed in 37°C for 90 min for Rhod123 exclusion in a dark compartment. Cells kept on ice to inhibit exclusion of Rhod123 were used as a positive control for gating in flow cytometry. Fractions of Rhod123(+) and Rhod123(–) cells were sorted using FACS Aria and a left panel shows a gating strategy. ( B ) Analysis of SPP1, OCT3/4 and NANOG gene expression in Rhod 123(–) subpopulations sorted from four human glioma cell lines. Total RNA was isolated from sorted cells using Qiagen RNeasy kit and the levels of SPP1, NANOG and OCT3/4 mRNA were determined by qPCR in Rhod 123(+) and Rhod 123(–) subpopulations; their expression in Rhod 123(+) subpopulations was taken as 1 (a red line). P values were calculated with a t-test and considered significant when *P ≤ 0.05 and * *P ≤ 0.01. ( C ) Quantification of the expression of Spp1, Oct3/4 and Nanog in Rhod 123(–) subpopulations isolated from rat C6 glioma cells versus their levels in Rhod 123(+) subpopulations taken as 1 (a red line); n = 3.

    Article Snippet: Real- time PCR Total RNA was isolated from glioma cells using Qiagen RNeasy kit, 1 μg was used as a template.

    Techniques: Expressing, Flow Cytometry, Cytometry, Incubation, Positive Control, FACS, Isolation, Real-time Polymerase Chain Reaction

    Alterations in cardiac microRNA expression 16–26 days after percutaneous cardiac delivery of scAAV6-U6-shRNA-PLB ( n =3) compared with control ( n =3). Microarray analysis revealed significant alterations in expression of two miRNAs after overexpression of shRNA. No significant changes were noted in several other miRNAs with previously defined roles in the heart. *Significant, as defined by p

    Journal: Human Gene Therapy

    Article Title: Cardiac Gene Transfer of Short Hairpin RNA Directed Against Phospholamban Effectively Knocks Down Gene Expression but Causes Cellular Toxicity in Canines

    doi: 10.1089/hum.2011.035

    Figure Lengend Snippet: Alterations in cardiac microRNA expression 16–26 days after percutaneous cardiac delivery of scAAV6-U6-shRNA-PLB ( n =3) compared with control ( n =3). Microarray analysis revealed significant alterations in expression of two miRNAs after overexpression of shRNA. No significant changes were noted in several other miRNAs with previously defined roles in the heart. *Significant, as defined by p

    Article Snippet: MicroRNA (miRNA) was extracted with an miRNA Easy mini kit (Qiagen). miRNA expression was analyzed with miRCURY LNA version 9.2 (Exiqon, Woburn, MA).

    Techniques: Expressing, shRNA, Microarray, Over Expression