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Agilent technologies rna
Bias in miRNA detection using various <t>small-RNA</t> library preparation kits. For each kit, sequencing libraries were prepared from the miRXplore™ pool and sequenced; the sequence data were then used to calculate fold-deviations from the equimolar input and plotted as log 2 values. Densities of miRNAs within a two-fold deviation from the expected values (between vertical lines ) are considered unbiased according to [ 8 ]. Under-represented, over-represented, and accurately quantified percentages of miRNAs are shown in red font . Results for two-adapter schemes are a <t>TruSeq®</t> Small RNA, b NEBNext®, and c QIAseq. d NEXTFlex™, a scheme using two adapters with randomized sequences. e SMARTer, which uses template switching. f RealSeq®-AC, which uses a single-adapter and circularization (* p value vs other kits
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1) Product Images from "Decreasing miRNA sequencing bias using a single adapter and circularization approach"

Article Title: Decreasing miRNA sequencing bias using a single adapter and circularization approach

Journal: Genome Biology

doi: 10.1186/s13059-018-1488-z

Bias in miRNA detection using various small-RNA library preparation kits. For each kit, sequencing libraries were prepared from the miRXplore™ pool and sequenced; the sequence data were then used to calculate fold-deviations from the equimolar input and plotted as log 2 values. Densities of miRNAs within a two-fold deviation from the expected values (between vertical lines ) are considered unbiased according to [ 8 ]. Under-represented, over-represented, and accurately quantified percentages of miRNAs are shown in red font . Results for two-adapter schemes are a TruSeq® Small RNA, b NEBNext®, and c QIAseq. d NEXTFlex™, a scheme using two adapters with randomized sequences. e SMARTer, which uses template switching. f RealSeq®-AC, which uses a single-adapter and circularization (* p value vs other kits
Figure Legend Snippet: Bias in miRNA detection using various small-RNA library preparation kits. For each kit, sequencing libraries were prepared from the miRXplore™ pool and sequenced; the sequence data were then used to calculate fold-deviations from the equimolar input and plotted as log 2 values. Densities of miRNAs within a two-fold deviation from the expected values (between vertical lines ) are considered unbiased according to [ 8 ]. Under-represented, over-represented, and accurately quantified percentages of miRNAs are shown in red font . Results for two-adapter schemes are a TruSeq® Small RNA, b NEBNext®, and c QIAseq. d NEXTFlex™, a scheme using two adapters with randomized sequences. e SMARTer, which uses template switching. f RealSeq®-AC, which uses a single-adapter and circularization (* p value vs other kits

Techniques Used: Sequencing

Differential quantification of brain samples between different small RNA library preparation kits. Data obtained with either a TruSeq®, b NEBNext®, c NEXTFlex™, d QIAseq, or e SMARTer kits were compared with data obtained with RealSeq®-AC to obtain differential quantification (log 2 ) values for 276 high-confidence miRNAs. These values were plotted against the accuracy of detection of that miRNA when profiling the equimolar pool of synthetic miRNAs (Fig. 2 a–c). f–j The reverse comparison, with the differential quantification of RealSeq®-AC versus each of the other kits plotted against the accuracy of RealSeq®-AC when quantifying the synthetic pool of miRNAs. FN false negative, FP false positive. See Methods for more details
Figure Legend Snippet: Differential quantification of brain samples between different small RNA library preparation kits. Data obtained with either a TruSeq®, b NEBNext®, c NEXTFlex™, d QIAseq, or e SMARTer kits were compared with data obtained with RealSeq®-AC to obtain differential quantification (log 2 ) values for 276 high-confidence miRNAs. These values were plotted against the accuracy of detection of that miRNA when profiling the equimolar pool of synthetic miRNAs (Fig. 2 a–c). f–j The reverse comparison, with the differential quantification of RealSeq®-AC versus each of the other kits plotted against the accuracy of RealSeq®-AC when quantifying the synthetic pool of miRNAs. FN false negative, FP false positive. See Methods for more details

Techniques Used:

2) Product Images from "Tumour-vasculature development via endothelial-to-mesenchymal transition after radiotherapy controls CD44v6+ cancer cell and macrophage polarization"

Article Title: Tumour-vasculature development via endothelial-to-mesenchymal transition after radiotherapy controls CD44v6+ cancer cell and macrophage polarization

Journal: Nature Communications

doi: 10.1038/s41467-018-07470-w

TRP53-regulated EndMT modulates the M1 and M2 populations of increased TAMs after radiotherapy. a RNA-seq data analysis of the samples represented in Fig. 4a . Venn diagram depicting differentially expressed genes in irradiated HUVECs. Reverse-regulated genes ( > 1.2-fold) between TGFβR2 knockdown+IR and Trp53 -knockdown (with and without TGFβR2 knockdown)+IR conditions (compared with IR alone) were selected (subgroup A). Only matrisome- and surface-associated genes in subset A are shown in the heatmap generated by k-means clustering (subgroup B). b GeneMANIA network analysis of subset B showing significant enrichment for the GO term ‘leukocyte migration’. Red-filled circles represent genes upregulated in the Trp53 siRNA+IR group vs. the IR-alone group. Blue-filled circles represent genes upregulated in the TGFβR2 siRNA+IR group vs. the IR-alone group. The names of leukocyte migration-related genes are indicated in red. c Immunofluorescence detection of F4/80, CD31, and Arg1, or iNOS, CD31, and F4/80 in KP tumours from WT and EC-p53KO mice, with or without irradiation (23 days after irradiation). Scale bar = 20 μm. d Quantification of F4/80 + Arg1 + and F4/80 + iNOS + cells/field (magnification, ×200) using immunofluorescence images of KP tumours from WT, EC-p53KO, and TGFβR2KD mice, with or without irradiation (23 days after irradiation). e Ratio of CD206 + in F4/80 + cells from WT and EC-p53KO tumours 7 days after irradiation, as determined by flow cytometry. f Immunofluorescence staining of F4/80 and Arg1 in WT bone marrow-derived macrophages after coculture with KP tumour-derived ECs from WT and EC-p53KO mice for 48 h after irradiation. Scale bar = 50 μm (crop, 20 μm). Flow-cytometric analysis of CD206 + , iNOS + , and EdU + cells among F4/80 + cells (magnification, ×200) is shown. For ( d – f ), error bars indicate SEM; * p
Figure Legend Snippet: TRP53-regulated EndMT modulates the M1 and M2 populations of increased TAMs after radiotherapy. a RNA-seq data analysis of the samples represented in Fig. 4a . Venn diagram depicting differentially expressed genes in irradiated HUVECs. Reverse-regulated genes ( > 1.2-fold) between TGFβR2 knockdown+IR and Trp53 -knockdown (with and without TGFβR2 knockdown)+IR conditions (compared with IR alone) were selected (subgroup A). Only matrisome- and surface-associated genes in subset A are shown in the heatmap generated by k-means clustering (subgroup B). b GeneMANIA network analysis of subset B showing significant enrichment for the GO term ‘leukocyte migration’. Red-filled circles represent genes upregulated in the Trp53 siRNA+IR group vs. the IR-alone group. Blue-filled circles represent genes upregulated in the TGFβR2 siRNA+IR group vs. the IR-alone group. The names of leukocyte migration-related genes are indicated in red. c Immunofluorescence detection of F4/80, CD31, and Arg1, or iNOS, CD31, and F4/80 in KP tumours from WT and EC-p53KO mice, with or without irradiation (23 days after irradiation). Scale bar = 20 μm. d Quantification of F4/80 + Arg1 + and F4/80 + iNOS + cells/field (magnification, ×200) using immunofluorescence images of KP tumours from WT, EC-p53KO, and TGFβR2KD mice, with or without irradiation (23 days after irradiation). e Ratio of CD206 + in F4/80 + cells from WT and EC-p53KO tumours 7 days after irradiation, as determined by flow cytometry. f Immunofluorescence staining of F4/80 and Arg1 in WT bone marrow-derived macrophages after coculture with KP tumour-derived ECs from WT and EC-p53KO mice for 48 h after irradiation. Scale bar = 50 μm (crop, 20 μm). Flow-cytometric analysis of CD206 + , iNOS + , and EdU + cells among F4/80 + cells (magnification, ×200) is shown. For ( d – f ), error bars indicate SEM; * p

Techniques Used: RNA Sequencing Assay, Irradiation, Generated, Migration, Immunofluorescence, Mouse Assay, Flow Cytometry, Cytometry, Staining, Derivative Assay

Increased OPN expression during radiation-induced EndMT correlates with proliferating CD44v6 + cancer cells in hypoxic areas. a – c HUVECs were transfected with siRNAs targeting TGFβR2 , Trp53 , or TGF β R2 + Trp53 for 48 h and irradiated (10 Gy). After 48 h in culture, total RNA was isolated for RNA-seq and RT-qPCR analyses. a Heat map of RNA-seq analysis showing inhibition of radiation-induced EndMT and the mesenchymal phenotype in HUVECs by Trp53 knockdown. b RT-qPCR analysis of EC-adhesion molecules, collagen, and fibroblastic markers in HUVECs. c RT-qPCR analysis of OPN in HUVECs. d Human soluble-receptor array analysis of conditioned medium from human pulmonary microvascular endothelial cells at 8 days after 5 Gy irradiation. The cyan boxes mark the spots corresponding to OPN. e , f Immunofluorescence detection of CD31 and OPN ( e ) or CD44v6, OPN, and CD31 ( f ) in KP tumours from irradiated WT and EC-p53KO mice (23 dpi). Scale bar = 20 μm. g Quantification of OPN + αSMA + CD31 + cells in the total CD31 + area (left) and OPN + CD44v6 + cells in the total CD44v6 + area (right) (magnification, ×200; n > 5). Mouse strain designations are the same as in Figs 1 and 2 . IR irradiation (20 Gy). h , i KP tumour cells were cultured under normoxia, 1% O 2 (mild hypoxia), or 0.5% O 2 (severe hypoxia) and irradiated with or without OPN. Data are representative of three independent experiments. h Immunofluorescence detection of CD44v6 and Ki67 in KP tumour cells 11 days after irradiation (left) and quantification of CD44v6 + Ki67 + cells in total CD44v6 + cells (right) (magnification, ×200; n > 5). Scale bar = 50 μm. i Flow-cytometric analysis of EdU incorporation and CD44v6 expression in KP tumour cells 11 days after irradiation. For ( b – d ), the error bars indicate SD of three independent experiments. For ( g – i ), the error bars indicate SEM; For ( b, c ), si-TGFβR2 + IR or si-Control + IR versus si-Control group, # p
Figure Legend Snippet: Increased OPN expression during radiation-induced EndMT correlates with proliferating CD44v6 + cancer cells in hypoxic areas. a – c HUVECs were transfected with siRNAs targeting TGFβR2 , Trp53 , or TGF β R2 + Trp53 for 48 h and irradiated (10 Gy). After 48 h in culture, total RNA was isolated for RNA-seq and RT-qPCR analyses. a Heat map of RNA-seq analysis showing inhibition of radiation-induced EndMT and the mesenchymal phenotype in HUVECs by Trp53 knockdown. b RT-qPCR analysis of EC-adhesion molecules, collagen, and fibroblastic markers in HUVECs. c RT-qPCR analysis of OPN in HUVECs. d Human soluble-receptor array analysis of conditioned medium from human pulmonary microvascular endothelial cells at 8 days after 5 Gy irradiation. The cyan boxes mark the spots corresponding to OPN. e , f Immunofluorescence detection of CD31 and OPN ( e ) or CD44v6, OPN, and CD31 ( f ) in KP tumours from irradiated WT and EC-p53KO mice (23 dpi). Scale bar = 20 μm. g Quantification of OPN + αSMA + CD31 + cells in the total CD31 + area (left) and OPN + CD44v6 + cells in the total CD44v6 + area (right) (magnification, ×200; n > 5). Mouse strain designations are the same as in Figs 1 and 2 . IR irradiation (20 Gy). h , i KP tumour cells were cultured under normoxia, 1% O 2 (mild hypoxia), or 0.5% O 2 (severe hypoxia) and irradiated with or without OPN. Data are representative of three independent experiments. h Immunofluorescence detection of CD44v6 and Ki67 in KP tumour cells 11 days after irradiation (left) and quantification of CD44v6 + Ki67 + cells in total CD44v6 + cells (right) (magnification, ×200; n > 5). Scale bar = 50 μm. i Flow-cytometric analysis of EdU incorporation and CD44v6 expression in KP tumour cells 11 days after irradiation. For ( b – d ), the error bars indicate SD of three independent experiments. For ( g – i ), the error bars indicate SEM; For ( b, c ), si-TGFβR2 + IR or si-Control + IR versus si-Control group, # p

Techniques Used: Expressing, Transfection, Irradiation, Isolation, RNA Sequencing Assay, Quantitative RT-PCR, Inhibition, Immunofluorescence, Mouse Assay, Cell Culture, Flow Cytometry

3) Product Images from "Intratumoural Heterogeneity Underlies Distinct Therapy Responses and Treatment Resistance in Glioblastoma"

Article Title: Intratumoural Heterogeneity Underlies Distinct Therapy Responses and Treatment Resistance in Glioblastoma

Journal: Cancers

doi: 10.3390/cancers11020190

Comprehensive genomic analyses of single-cell clones. ( A ) Genomic proportion of copy number alteration. For clonal samples B–F, > 84% of their genome is between copy number 3 and 4 (ranging from 84.82% to 85.9%, shown as dark grey bars) as compared to ≤3% for the other samples. This global whole genome duplication typically affected both alleles, preserving heterozygosity in these samples. ( B ) Number of somatic structural variants identified per sample with the type indicated by colours. ( C ) Hierarchical clustering of somatic substitution variant allele frequencies across tumour and clonal samples. The absence of a variant in the normal control cortex sample is demonstrated by a variant allele frequency of zero (dark blue). ( D ) Hierarchical clustering of Log2-normalised and gene-scaled RNA seq gene expression of genes with most variable expression as identified as contributing to the first principle component. Positive values indicate a sample with the highest expression and negative values with the lowest expression. ( E ) Hierarchical clustering of 1000 most variable β-values from DNA methylation sequencing data. Β-value of 1 indicates completely methylated and 0 unmethylated CpGs.
Figure Legend Snippet: Comprehensive genomic analyses of single-cell clones. ( A ) Genomic proportion of copy number alteration. For clonal samples B–F, > 84% of their genome is between copy number 3 and 4 (ranging from 84.82% to 85.9%, shown as dark grey bars) as compared to ≤3% for the other samples. This global whole genome duplication typically affected both alleles, preserving heterozygosity in these samples. ( B ) Number of somatic structural variants identified per sample with the type indicated by colours. ( C ) Hierarchical clustering of somatic substitution variant allele frequencies across tumour and clonal samples. The absence of a variant in the normal control cortex sample is demonstrated by a variant allele frequency of zero (dark blue). ( D ) Hierarchical clustering of Log2-normalised and gene-scaled RNA seq gene expression of genes with most variable expression as identified as contributing to the first principle component. Positive values indicate a sample with the highest expression and negative values with the lowest expression. ( E ) Hierarchical clustering of 1000 most variable β-values from DNA methylation sequencing data. Β-value of 1 indicates completely methylated and 0 unmethylated CpGs.

Techniques Used: Clone Assay, Preserving, Variant Assay, RNA Sequencing Assay, Expressing, DNA Methylation Assay, Sequencing, Methylation

4) Product Images from "Biobanking: Objectives, Requirements, and Future Challenges—Experiences from the Munich Vascular Biobank"

Article Title: Biobanking: Objectives, Requirements, and Future Challenges—Experiences from the Munich Vascular Biobank

Journal: Journal of Clinical Medicine

doi: 10.3390/jcm8020251

Evaluation of the extent of RNA fragmentation from FFPE tissue samples measuring the area under the curve from Agilent Bioanalyzer. Two values were defined: maximal RNA length and the 50% RNA length calculated as a 50% reduction of the area under the curve. RIN: RNA integrity number; FFPE: formalin-fixed paraffin-embedded specimens. nt: number of nucleotides; FU: fluorescence unit.
Figure Legend Snippet: Evaluation of the extent of RNA fragmentation from FFPE tissue samples measuring the area under the curve from Agilent Bioanalyzer. Two values were defined: maximal RNA length and the 50% RNA length calculated as a 50% reduction of the area under the curve. RIN: RNA integrity number; FFPE: formalin-fixed paraffin-embedded specimens. nt: number of nucleotides; FU: fluorescence unit.

Techniques Used: Formalin-fixed Paraffin-Embedded, Fluorescence

( A ) Measurement of RNA integrity number (RIN) in FFPE vascular tissue samples using Agilent Bioanalyzer between 2009 and 2018 ( n = 5 for each group and year). ( B ) Evaluation of the length of the RNA fragments, as described in Figure 3 . No significant differences were observed between the study years over time.
Figure Legend Snippet: ( A ) Measurement of RNA integrity number (RIN) in FFPE vascular tissue samples using Agilent Bioanalyzer between 2009 and 2018 ( n = 5 for each group and year). ( B ) Evaluation of the length of the RNA fragments, as described in Figure 3 . No significant differences were observed between the study years over time.

Techniques Used: Formalin-fixed Paraffin-Embedded

5) Product Images from "Laser microdissection coupled with RNA-seq analysis of porcine enterocytes infected with an obligate intracellular pathogen (Lawsonia intracellularis)"

Article Title: Laser microdissection coupled with RNA-seq analysis of porcine enterocytes infected with an obligate intracellular pathogen (Lawsonia intracellularis)

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-421

Correlation between RNA-seq and qRT-PCR expression data. (A) Plot of the relative fold-change in gene expression of 16 porcine genes from infected enterocytes by RNA-seq (x-axis) and qRT-PCR (y-axis). R 2 = 0.92 ( p
Figure Legend Snippet: Correlation between RNA-seq and qRT-PCR expression data. (A) Plot of the relative fold-change in gene expression of 16 porcine genes from infected enterocytes by RNA-seq (x-axis) and qRT-PCR (y-axis). R 2 = 0.92 ( p

Techniques Used: RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Infection

Laser capture microdissection of an intestinal crypt infected with L. intracellularis (A-B) and evaluation of RNA quality from microdissected cells (C-D). (A) Hematoxylin stained cryosection of infected ileal mucosa. (B) Microdissected intestinal crypt captured in the thermoplastic film of the LCM cap prior to RNA isolation. (C) Agilent Bioanalyzer data showing bacterial and eukaryotic ribosomal RNA in infected and non-infected cells. (D) One-step RT-PCR products of three protein-encoding genes of L. intracellularis .
Figure Legend Snippet: Laser capture microdissection of an intestinal crypt infected with L. intracellularis (A-B) and evaluation of RNA quality from microdissected cells (C-D). (A) Hematoxylin stained cryosection of infected ileal mucosa. (B) Microdissected intestinal crypt captured in the thermoplastic film of the LCM cap prior to RNA isolation. (C) Agilent Bioanalyzer data showing bacterial and eukaryotic ribosomal RNA in infected and non-infected cells. (D) One-step RT-PCR products of three protein-encoding genes of L. intracellularis .

Techniques Used: Laser Capture Microdissection, Infection, Staining, Isolation, Reverse Transcription Polymerase Chain Reaction

6) Product Images from "Characterization of human plasma-derived exosomal RNAs by deep sequencing"

Article Title: Characterization of human plasma-derived exosomal RNAs by deep sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-319

Other RNA species that were detected in the exosomal RNA libraries. ( A ) Pie chart of RNA species and their distributions in the plasma-derived exosomes. Misc RNAs are the RNA sequences that mapped to the human genome but not in any of the categories listed. The DNA category represents the novel transcripts that have no annotation in the human RNA database. ( B ) Graphic and statistics of a representative novel miRNA predicted by miRDeep2. Both star and mature strands were detected and integrated. Lower left table shows information about the sample and the miRDeep2 scores, along with the read count for each component of the putative miRNA. mm, number of mismatches. Mismatched nucleotides are indicated by uppercase letters.
Figure Legend Snippet: Other RNA species that were detected in the exosomal RNA libraries. ( A ) Pie chart of RNA species and their distributions in the plasma-derived exosomes. Misc RNAs are the RNA sequences that mapped to the human genome but not in any of the categories listed. The DNA category represents the novel transcripts that have no annotation in the human RNA database. ( B ) Graphic and statistics of a representative novel miRNA predicted by miRDeep2. Both star and mature strands were detected and integrated. Lower left table shows information about the sample and the miRDeep2 scores, along with the read count for each component of the putative miRNA. mm, number of mismatches. Mismatched nucleotides are indicated by uppercase letters.

Techniques Used: Derivative Assay

Library preparation and analysis of the raw sequencing data. ( A ) PAGE analysis of the sequencing libraries prepared using three different kits. 20-bp DNA ladders are shown on the left of each panel while the prepared libraries are shown on the right. The anticipated RNA sequencing constructs ranging from 140–160 bp are highlighted within brackets. The DNA bands (~125 bp) are the adaptor dimers. ( B ) Percentage of mappable RNAs in the raw sequencing reads. ( C ) Abundance of the selected miRNAs and β-actin in plasma exosomal RNA and HEK293 cellular RNA. Gene and miRNA abundance was determined by the threshold cycle (Ct), where Ct values > 30 are defined as rare. ( D ) Percentage of each miRNA in the repertoire of total miRNA reads.
Figure Legend Snippet: Library preparation and analysis of the raw sequencing data. ( A ) PAGE analysis of the sequencing libraries prepared using three different kits. 20-bp DNA ladders are shown on the left of each panel while the prepared libraries are shown on the right. The anticipated RNA sequencing constructs ranging from 140–160 bp are highlighted within brackets. The DNA bands (~125 bp) are the adaptor dimers. ( B ) Percentage of mappable RNAs in the raw sequencing reads. ( C ) Abundance of the selected miRNAs and β-actin in plasma exosomal RNA and HEK293 cellular RNA. Gene and miRNA abundance was determined by the threshold cycle (Ct), where Ct values > 30 are defined as rare. ( D ) Percentage of each miRNA in the repertoire of total miRNA reads.

Techniques Used: Sequencing, Polyacrylamide Gel Electrophoresis, RNA Sequencing Assay, Construct

Exosome isolation and exosomal small RNA quantification. ( A ) Representative histogram of exosome size distribution. ( B ) Exosomal RNAs determined using the Agilent Small RNA Chip. The small RNAs were dominant in the exosomal RNAs. ( C ) Exosomal RNAs determined by the Agilent RNA Pico Chip. Exosomal RNA samples A , B and C contained no detectable 18S and 28S rRNAs. Cellular RNA from HEK293 was loaded as positive control for 18S and 28S rRNAs ( D ) Isolated exosomal RNAs treated either with DNase I or RNase A. ( E ) Plasma and control small RNAs treated under various conditions.
Figure Legend Snippet: Exosome isolation and exosomal small RNA quantification. ( A ) Representative histogram of exosome size distribution. ( B ) Exosomal RNAs determined using the Agilent Small RNA Chip. The small RNAs were dominant in the exosomal RNAs. ( C ) Exosomal RNAs determined by the Agilent RNA Pico Chip. Exosomal RNA samples A , B and C contained no detectable 18S and 28S rRNAs. Cellular RNA from HEK293 was loaded as positive control for 18S and 28S rRNAs ( D ) Isolated exosomal RNAs treated either with DNase I or RNase A. ( E ) Plasma and control small RNAs treated under various conditions.

Techniques Used: Isolation, Chromatin Immunoprecipitation, Positive Control

Exosome isolation and exosomal small RNA quantification. ( A ) Representative histogram of exosome size distribution. ( B ) Exosomal RNAs determined using the Agilent Small RNA Chip. The small RNAs were dominant in the exosomal RNAs. ( C ) Exosomal RNAs determined by the Agilent RNA Pico Chip. Exosomal RNA samples A , B and C contained no detectable 18S and 28S rRNAs. Cellular RNA from HEK293 was loaded as positive control for 18S and 28S rRNAs ( D ) Isolated exosomal RNAs treated either with DNase I or RNase A. ( E ) Plasma and control small RNAs treated under various conditions.
Figure Legend Snippet: Exosome isolation and exosomal small RNA quantification. ( A ) Representative histogram of exosome size distribution. ( B ) Exosomal RNAs determined using the Agilent Small RNA Chip. The small RNAs were dominant in the exosomal RNAs. ( C ) Exosomal RNAs determined by the Agilent RNA Pico Chip. Exosomal RNA samples A , B and C contained no detectable 18S and 28S rRNAs. Cellular RNA from HEK293 was loaded as positive control for 18S and 28S rRNAs ( D ) Isolated exosomal RNAs treated either with DNase I or RNase A. ( E ) Plasma and control small RNAs treated under various conditions.

Techniques Used: Isolation, Chromatin Immunoprecipitation, Positive Control

7) Product Images from "Characterization of human plasma-derived exosomal RNAs by deep sequencing"

Article Title: Characterization of human plasma-derived exosomal RNAs by deep sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-319

Exosome isolation and exosomal small RNA quantification. ( A ) Representative histogram of exosome size distribution. ( B ) Exosomal RNAs determined using the Agilent Small RNA Chip. The small RNAs were dominant in the exosomal RNAs. ( C ) Exosomal RNAs determined by the Agilent RNA Pico Chip. Exosomal RNA samples A , B and C contained no detectable 18S and 28S rRNAs. Cellular RNA from HEK293 was loaded as positive control for 18S and 28S rRNAs ( D ) Isolated exosomal RNAs treated either with DNase I or RNase A. ( E ) Plasma and control small RNAs treated under various conditions.
Figure Legend Snippet: Exosome isolation and exosomal small RNA quantification. ( A ) Representative histogram of exosome size distribution. ( B ) Exosomal RNAs determined using the Agilent Small RNA Chip. The small RNAs were dominant in the exosomal RNAs. ( C ) Exosomal RNAs determined by the Agilent RNA Pico Chip. Exosomal RNA samples A , B and C contained no detectable 18S and 28S rRNAs. Cellular RNA from HEK293 was loaded as positive control for 18S and 28S rRNAs ( D ) Isolated exosomal RNAs treated either with DNase I or RNase A. ( E ) Plasma and control small RNAs treated under various conditions.

Techniques Used: Isolation, Chromatin Immunoprecipitation, Positive Control

8) Product Images from "A qualitative assessment of direct-labeled cDNA products prior to microarray analysis"

Article Title: A qualitative assessment of direct-labeled cDNA products prior to microarray analysis

Journal: BMC Genomics

doi: 10.1186/1471-2164-6-36

RNA and Cy5-dUTP labeled cDNA measured by the Agilent 2100 Bioanalyzer. Electropherogram images (one representative experiment) of RNAs treated with RNase as follows: No RNase (A, pink), 0.01 ng ml-1 (B, blue), 0.1 ng ml-1 (C, red), 1 ng ml-1 (D, green). Overlayed electropherogram image of Cy5-dUTP labeled cDNA (E, colors as described above, one representative experiment). The first peak (as shown by an arrow) from the left in all electropherograms is a 50 base pair marker present in the sample buffer (M). The second peak (as shown by an arrow) that is present only in Cy5 samples is free Cy5-dUTP (Cy5) and measures approximately 150 nucleotides (data not shown). Though it is a single nucleotide, the Cy5 modification is large, which could explain its delayed migration and detection.
Figure Legend Snippet: RNA and Cy5-dUTP labeled cDNA measured by the Agilent 2100 Bioanalyzer. Electropherogram images (one representative experiment) of RNAs treated with RNase as follows: No RNase (A, pink), 0.01 ng ml-1 (B, blue), 0.1 ng ml-1 (C, red), 1 ng ml-1 (D, green). Overlayed electropherogram image of Cy5-dUTP labeled cDNA (E, colors as described above, one representative experiment). The first peak (as shown by an arrow) from the left in all electropherograms is a 50 base pair marker present in the sample buffer (M). The second peak (as shown by an arrow) that is present only in Cy5 samples is free Cy5-dUTP (Cy5) and measures approximately 150 nucleotides (data not shown). Though it is a single nucleotide, the Cy5 modification is large, which could explain its delayed migration and detection.

Techniques Used: Labeling, Marker, Modification, Migration

9) Product Images from "Gene Expression Profiling during Conidiation in the Rice Blast Pathogen Magnaporthe oryzae"

Article Title: Gene Expression Profiling during Conidiation in the Rice Blast Pathogen Magnaporthe oryzae

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043202

The genome-wide analysis of changes in mRNA abundance during conidiation. ( A ). Measurement of the conidial density of M. oryzae on polycarbonate membrane-laid OMA plates at the indicated times. ( B ). The number of genes induced (left) or repressed (right) during conidiation based on the comparison of RNA levels of non-conidiating mycelia (NCMY) with conidiating mycelia (CNMY) of M. oryzae . ( C ). The number of genes induced (right) or repressed (left) with fold change values during conidiation of M. oryzae . ( D ). Validation of the microarray data by qRT-PCR. Transcript levels of each gene in CNMY were normalized to β -tubulin and expressed as relative values, with 1 corresponding to the NCMY. Each of five-digit number on the x-axis indicates the MGG locus number.
Figure Legend Snippet: The genome-wide analysis of changes in mRNA abundance during conidiation. ( A ). Measurement of the conidial density of M. oryzae on polycarbonate membrane-laid OMA plates at the indicated times. ( B ). The number of genes induced (left) or repressed (right) during conidiation based on the comparison of RNA levels of non-conidiating mycelia (NCMY) with conidiating mycelia (CNMY) of M. oryzae . ( C ). The number of genes induced (right) or repressed (left) with fold change values during conidiation of M. oryzae . ( D ). Validation of the microarray data by qRT-PCR. Transcript levels of each gene in CNMY were normalized to β -tubulin and expressed as relative values, with 1 corresponding to the NCMY. Each of five-digit number on the x-axis indicates the MGG locus number.

Techniques Used: Genome Wide, Microarray, Quantitative RT-PCR

The genome-wide analysis of changes in mRNA abundance in the Δ Mohox2 mutant during conidiation. ( A ). The number of genes induced (left) or repressed (right) with fold change values by comparing RNA levels in wild type with those in the Δ Mohox2 mutant during conidiation. ( B ). The number of genes induced in wild-type and repressed in the Δ Mohox2 mutant during conidiation. ( C ). Validation of microarray data by qRT-PCR. Graph shows the transcript levels of each gene on the same x-axis in the wild-type (white squares) and in the Δ Mohox2 mutant (black squares) during conidiation.
Figure Legend Snippet: The genome-wide analysis of changes in mRNA abundance in the Δ Mohox2 mutant during conidiation. ( A ). The number of genes induced (left) or repressed (right) with fold change values by comparing RNA levels in wild type with those in the Δ Mohox2 mutant during conidiation. ( B ). The number of genes induced in wild-type and repressed in the Δ Mohox2 mutant during conidiation. ( C ). Validation of microarray data by qRT-PCR. Graph shows the transcript levels of each gene on the same x-axis in the wild-type (white squares) and in the Δ Mohox2 mutant (black squares) during conidiation.

Techniques Used: Genome Wide, Mutagenesis, Microarray, Quantitative RT-PCR

10) Product Images from "Expression and characterization of a ?-fructofuranosidase from the parasitic protist Trichomonas vaginalis"

Article Title: Expression and characterization of a ?-fructofuranosidase from the parasitic protist Trichomonas vaginalis

Journal: BMC Biochemistry

doi: 10.1186/1471-2091-15-12

Amplification of the TVAG_254130 open reading frame from T. vaginalis cDNA. Total RNA was isolated from a 40 hr culture of T. vaginalis grown under our standard conditions. The resulting RNA was used to synthesize cDNA (+RT). To control for genomic DNA contamination, mock reactions were carried out in which reverse transcriptase was omitted from the cDNA synthesis step (-RT).
Figure Legend Snippet: Amplification of the TVAG_254130 open reading frame from T. vaginalis cDNA. Total RNA was isolated from a 40 hr culture of T. vaginalis grown under our standard conditions. The resulting RNA was used to synthesize cDNA (+RT). To control for genomic DNA contamination, mock reactions were carried out in which reverse transcriptase was omitted from the cDNA synthesis step (-RT).

Techniques Used: Amplification, Isolation

11) Product Images from "Optimization of laser capture microdissection and RNA amplification for gene expression profiling of prostate cancer"

Article Title: Optimization of laser capture microdissection and RNA amplification for gene expression profiling of prostate cancer

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-8-25

Yield and integrity of RNA isolated from laser capture microdissected cells . (Left axis), 3'/M beta-actin ratios determined by qPCR as a measure of the quality of RNA samples obtained by LCM of a total of 27 cases of benign prostate, benign prostatic hyperplasia (BPH), primary Gleason pattern 3 (GP3), pattern 4 (GP4), pattern 5 (GP5), and metastatic prostate cancer (Met). (Right axis), relative yields of LCM RNA samples based on qPCR with primers specific for the 3' end of the beta-actin transcript.
Figure Legend Snippet: Yield and integrity of RNA isolated from laser capture microdissected cells . (Left axis), 3'/M beta-actin ratios determined by qPCR as a measure of the quality of RNA samples obtained by LCM of a total of 27 cases of benign prostate, benign prostatic hyperplasia (BPH), primary Gleason pattern 3 (GP3), pattern 4 (GP4), pattern 5 (GP5), and metastatic prostate cancer (Met). (Right axis), relative yields of LCM RNA samples based on qPCR with primers specific for the 3' end of the beta-actin transcript.

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Laser Capture Microdissection

12) Product Images from "Selective amplification of Brucella melitensis mRNA from a mixed host-pathogen total RNA"

Article Title: Selective amplification of Brucella melitensis mRNA from a mixed host-pathogen total RNA

Journal: BMC Research Notes

doi: 10.1186/1756-0500-3-244

Integrity and composition of the host: Brucella RNA samples pre- and post-treatment . Twenty-five (25) μg total eukaryote RNA from human (HeLa S3) and bovine (MDBK) cell lines were mixed with 2 μg of Brucella melitensis 16 M RNA (ratio 12.5:1) and treated with MICROB Enrich ™(Ambion) according to the instruction manual. (A) Agarose gel electrophoresis image. Lines 1 2: HeLa cell line: B.melitensis 16 M RNA mix; lines 3 4: MDBK cell line: B.melitensis 16 M RNA mix; pre and post-treatment respectively. (B) Comparison of RNA composition from a sample of MDBK cell line: B. melitensis 16 M pre and post-treatment with MICROB Enrich ™ (Ambion) and examined on an Agilent 2100 Bioanalyzer. Ribosomal RNA subunits are indicated.
Figure Legend Snippet: Integrity and composition of the host: Brucella RNA samples pre- and post-treatment . Twenty-five (25) μg total eukaryote RNA from human (HeLa S3) and bovine (MDBK) cell lines were mixed with 2 μg of Brucella melitensis 16 M RNA (ratio 12.5:1) and treated with MICROB Enrich ™(Ambion) according to the instruction manual. (A) Agarose gel electrophoresis image. Lines 1 2: HeLa cell line: B.melitensis 16 M RNA mix; lines 3 4: MDBK cell line: B.melitensis 16 M RNA mix; pre and post-treatment respectively. (B) Comparison of RNA composition from a sample of MDBK cell line: B. melitensis 16 M pre and post-treatment with MICROB Enrich ™ (Ambion) and examined on an Agilent 2100 Bioanalyzer. Ribosomal RNA subunits are indicated.

Techniques Used: Agarose Gel Electrophoresis

13) Product Images from "Comparison of target labeling methods for use with Affymetrix GeneChips"

Article Title: Comparison of target labeling methods for use with Affymetrix GeneChips

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-7-24

Overlaid Bioanalyzer electropherograms for fragmented labeled cRNA targets showing the size distribution of fragmented target. One-Cycle replicates (blue and green); Superscript replicates (orange and black); BioArray replicates (pink and turquoise). RNA ladder in red showing the lower alignment marker and the 200 and 500 base markers.
Figure Legend Snippet: Overlaid Bioanalyzer electropherograms for fragmented labeled cRNA targets showing the size distribution of fragmented target. One-Cycle replicates (blue and green); Superscript replicates (orange and black); BioArray replicates (pink and turquoise). RNA ladder in red showing the lower alignment marker and the 200 and 500 base markers.

Techniques Used: Labeling, Marker

Overlaid electropherograms from the analysis of unfragmented biotinylated cRNA products from the IVT reactions of the 3 different labeling kits by the Agilent 2100 Bioanalyzer. The replicate reactions from donor A are shown for each kit: One-Cycle data represented as blue and green line; BioArray as black and orange and Superscript by the pink and turquoise lines. 1 μl of the final volume (One-Cycle = 21 μl; BioArray = 60 μl; Superscript = 100 μl) of purified IVT reaction is loaded. The RNA ladder (peaks represented in red) contains a mixture of RNAs of known concentration and size (50 (lower marker) 200, 500, 1,000, 2,000, 4,000, and 6,000 bases from left to right).
Figure Legend Snippet: Overlaid electropherograms from the analysis of unfragmented biotinylated cRNA products from the IVT reactions of the 3 different labeling kits by the Agilent 2100 Bioanalyzer. The replicate reactions from donor A are shown for each kit: One-Cycle data represented as blue and green line; BioArray as black and orange and Superscript by the pink and turquoise lines. 1 μl of the final volume (One-Cycle = 21 μl; BioArray = 60 μl; Superscript = 100 μl) of purified IVT reaction is loaded. The RNA ladder (peaks represented in red) contains a mixture of RNAs of known concentration and size (50 (lower marker) 200, 500, 1,000, 2,000, 4,000, and 6,000 bases from left to right).

Techniques Used: Labeling, Purification, Concentration Assay, Marker

14) Product Images from "Characterization of Whole Blood Gene Expression Profiles as a Sequel to Globin mRNA Reduction in Patients with Sickle Cell Disease"

Article Title: Characterization of Whole Blood Gene Expression Profiles as a Sequel to Globin mRNA Reduction in Patients with Sickle Cell Disease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0006484

A. Quality of total RNA from PBMC, PAX and PAX-GR. The intact 18 and 28s ribosomal bands are shown in the electophoregram determined in agilent bioanalyzer. B. Quality of biotinylated cRNA from PBMC, PAX and PAX-GR. A predominant 700 bp peak represents the globin cRNA in the PAX samples. cRNA from PBMC and PAX-GR show a similar pattern of a smear of transcripts. 1. Red line - PAX. 2. Blue line - PAX- GR. 3. Green line - PBMC.
Figure Legend Snippet: A. Quality of total RNA from PBMC, PAX and PAX-GR. The intact 18 and 28s ribosomal bands are shown in the electophoregram determined in agilent bioanalyzer. B. Quality of biotinylated cRNA from PBMC, PAX and PAX-GR. A predominant 700 bp peak represents the globin cRNA in the PAX samples. cRNA from PBMC and PAX-GR show a similar pattern of a smear of transcripts. 1. Red line - PAX. 2. Blue line - PAX- GR. 3. Green line - PBMC.

Techniques Used:

15) Product Images from "Microarray analysis of RNA extracted from formalin-fixed, paraffin-embedded and matched fresh-frozen ovarian adenocarcinomas"

Article Title: Microarray analysis of RNA extracted from formalin-fixed, paraffin-embedded and matched fresh-frozen ovarian adenocarcinomas

Journal: BMC Medical Genomics

doi: 10.1186/1755-8794-2-23

Bioanalyzer profiles of total RNA (A) and cRNA (B) of matched FF and FFPE ovarian serous adenocarcinoma samples 3136, 3138, 3194, 3207 and 390 . The method used for RNA extraction (Qiagen, Agencourt, Ambion) is indicated for each sample type. The RNA Integrity Number (RIN) is shown next to each total RNA profile.
Figure Legend Snippet: Bioanalyzer profiles of total RNA (A) and cRNA (B) of matched FF and FFPE ovarian serous adenocarcinoma samples 3136, 3138, 3194, 3207 and 390 . The method used for RNA extraction (Qiagen, Agencourt, Ambion) is indicated for each sample type. The RNA Integrity Number (RIN) is shown next to each total RNA profile.

Techniques Used: Formalin-fixed Paraffin-Embedded, RNA Extraction

16) Product Images from "Chemopreventive and Therapeutic Activity of Dietary Blueberry against Estrogen-Mediated Breast Cancer"

Article Title: Chemopreventive and Therapeutic Activity of Dietary Blueberry against Estrogen-Mediated Breast Cancer

Journal: Journal of Agricultural and Food Chemistry

doi: 10.1021/jf403734j

Expression of miRNAs 18a and 34c in mammary tissues. The small RNA was isolated by mirVana microRNA kit and quantified by Bioanalyzer. qPCR analysis was performed using a TaqMan microRNA Reverse Triscription Kit and TaqMan gene-specific MicroRNA assays. Graph represents the average of four rats ± SE done in duplicates. Asterisk indicates significant difference from E 2 -treated control ( p = 0.0013 and 0.0033).
Figure Legend Snippet: Expression of miRNAs 18a and 34c in mammary tissues. The small RNA was isolated by mirVana microRNA kit and quantified by Bioanalyzer. qPCR analysis was performed using a TaqMan microRNA Reverse Triscription Kit and TaqMan gene-specific MicroRNA assays. Graph represents the average of four rats ± SE done in duplicates. Asterisk indicates significant difference from E 2 -treated control ( p = 0.0013 and 0.0033).

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

17) Product Images from "Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta 1"

Article Title: Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta 1

Journal: Applications in Plant Sciences

doi: 10.3732/apps.1400105

Examples of Agilent 2100 Bioanalyzer spectra of total RNA showing improvement with Options 1 and 2 compared to Option 3. Each graph shows the intensity of the peaks of the ribosomal RNA subunits: nuclear large-28S, small-18S, cytoplasmic, mitochondrial, and chloroplastic (smaller subunits). The electrophoretic gel for each sample is shown to the right indicating the subunit bands or degradation (i.e., smear). nt = number of estimated nucleotides based on ladder; FU = fluorescence unit (i.e., intensity of peak). (A) Degraded ribosomal RNA subunits of Canella winterana (L.) Gaertn. extracted with Option 3 that resulted in an estimation of 27 μg of RNA, but the subunits are degraded. (B) A second example using Option 3, Muntingia calabura L., also shows an inflated quantity reading with degraded subunits. (C) Canella winterana , extracted with Option 1, indicating peaks for intact ribosomal RNA subunits. (D) Muntingia calabura extracted with Option 2.
Figure Legend Snippet: Examples of Agilent 2100 Bioanalyzer spectra of total RNA showing improvement with Options 1 and 2 compared to Option 3. Each graph shows the intensity of the peaks of the ribosomal RNA subunits: nuclear large-28S, small-18S, cytoplasmic, mitochondrial, and chloroplastic (smaller subunits). The electrophoretic gel for each sample is shown to the right indicating the subunit bands or degradation (i.e., smear). nt = number of estimated nucleotides based on ladder; FU = fluorescence unit (i.e., intensity of peak). (A) Degraded ribosomal RNA subunits of Canella winterana (L.) Gaertn. extracted with Option 3 that resulted in an estimation of 27 μg of RNA, but the subunits are degraded. (B) A second example using Option 3, Muntingia calabura L., also shows an inflated quantity reading with degraded subunits. (C) Canella winterana , extracted with Option 1, indicating peaks for intact ribosomal RNA subunits. (D) Muntingia calabura extracted with Option 2.

Techniques Used: Fluorescence

18) Product Images from "Reprogramming of Yersinia from Virulent to Persistent Mode Revealed by Complex In Vivo RNA-seq Analysis"

Article Title: Reprogramming of Yersinia from Virulent to Persistent Mode Revealed by Complex In Vivo RNA-seq Analysis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004600

Y. pseudotuberculosis infection alters the bacterial composition of the cecum. (A) Representative Bioanalyzer 2100 electrographs and associated gel pictures for replicates of in vitro -derived RNA samples (grown at 26°C and 37°C), in vivo -derived samples of early (isolated from mouse cecal tissue 2 dpi) and persistent infection (isolated from mouse cecal tissue 42 dpi), and uninfected samples (isolated from uninfected mouse cecal tissue). (B) The number of reads mapping to 16S rRNA from different bacteria in non-depleted in vivo -derived samples. Data represent the mean ± SD of the two replicates for each sample group. (C) Relative abundance of different bacterial phyla in samples according to reads mapped to the 16SMicrobial database. The proportions are given as the percent of bacterial phyla identified in specific samples.
Figure Legend Snippet: Y. pseudotuberculosis infection alters the bacterial composition of the cecum. (A) Representative Bioanalyzer 2100 electrographs and associated gel pictures for replicates of in vitro -derived RNA samples (grown at 26°C and 37°C), in vivo -derived samples of early (isolated from mouse cecal tissue 2 dpi) and persistent infection (isolated from mouse cecal tissue 42 dpi), and uninfected samples (isolated from uninfected mouse cecal tissue). (B) The number of reads mapping to 16S rRNA from different bacteria in non-depleted in vivo -derived samples. Data represent the mean ± SD of the two replicates for each sample group. (C) Relative abundance of different bacterial phyla in samples according to reads mapped to the 16SMicrobial database. The proportions are given as the percent of bacterial phyla identified in specific samples.

Techniques Used: Infection, In Vitro, Derivative Assay, In Vivo, Isolation

19) Product Images from "Mechanisms Contributing to Differential Regulation of PAX3 Downstream Target Genes in Normal Human Epidermal Melanocytes versus Melanoma Cells"

Article Title: Mechanisms Contributing to Differential Regulation of PAX3 Downstream Target Genes in Normal Human Epidermal Melanocytes versus Melanoma Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0124154

PAX3/ PAX3 expression in a representative panel of melanocytes and melanoma cell lines. A) PAX3 protein levels relative to β-actin. Each sample was run in duplicate on separate gels and the density of each band was assessed by densitometric scanning using the GS-800 Calibrated Densitometer (Bio-Rad). The density of each PAX3 band was compared to that of β-actin for each lane on the same membrane and the fold change calculated and graphed. The error bars represent the standard deviation between duplicate samples. B) PAX3 mRNA expression in neonatal and adult normal human epidermal melanocytes and primary and metastatic melanoma cell lines. RT-qPCR was performed using RNA from neonatal (NHEM-n) and adult (NHEM-a (P) and NHEM-a (I)) human epidermal melanocytes as well as six primary (MM200, MM229, MM329, MM540, MM622 and WM115) and five metastatic (A2058, M14, SKMEL2, SKMEL5 and UACC62) melanoma cell lines. The level of PAX3 expression was calculated as fold-change relative to GAPDH expression levels. All PCRs were performed in triplicate and average values were used to calculate fold change over GAPDH for each sample. All samples were run in biological duplicates and the error bars represent the standard deviation of the biological replicates. C) Immunocytochemistry of neonatal and adult human epidermal melanocytes, five metastatic melanoma cell lines and six primary melanoma cell lines. Nuclear PAX3 protein expression (red) is observed in all melanoma cell lines and primary melanocytes. Cell nuclei are stained with DAPI (blue). The scale measures 100 μM. A negative control was included with each experimental run and proved negative in each instance.
Figure Legend Snippet: PAX3/ PAX3 expression in a representative panel of melanocytes and melanoma cell lines. A) PAX3 protein levels relative to β-actin. Each sample was run in duplicate on separate gels and the density of each band was assessed by densitometric scanning using the GS-800 Calibrated Densitometer (Bio-Rad). The density of each PAX3 band was compared to that of β-actin for each lane on the same membrane and the fold change calculated and graphed. The error bars represent the standard deviation between duplicate samples. B) PAX3 mRNA expression in neonatal and adult normal human epidermal melanocytes and primary and metastatic melanoma cell lines. RT-qPCR was performed using RNA from neonatal (NHEM-n) and adult (NHEM-a (P) and NHEM-a (I)) human epidermal melanocytes as well as six primary (MM200, MM229, MM329, MM540, MM622 and WM115) and five metastatic (A2058, M14, SKMEL2, SKMEL5 and UACC62) melanoma cell lines. The level of PAX3 expression was calculated as fold-change relative to GAPDH expression levels. All PCRs were performed in triplicate and average values were used to calculate fold change over GAPDH for each sample. All samples were run in biological duplicates and the error bars represent the standard deviation of the biological replicates. C) Immunocytochemistry of neonatal and adult human epidermal melanocytes, five metastatic melanoma cell lines and six primary melanoma cell lines. Nuclear PAX3 protein expression (red) is observed in all melanoma cell lines and primary melanocytes. Cell nuclei are stained with DAPI (blue). The scale measures 100 μM. A negative control was included with each experimental run and proved negative in each instance.

Techniques Used: Expressing, Standard Deviation, Quantitative RT-PCR, Immunocytochemistry, Staining, Negative Control

20) Product Images from "Transcriptome-wide discovery of circular RNAs in Archaea"

Article Title: Transcriptome-wide discovery of circular RNAs in Archaea

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr1009

Identification of circular RNA products in RNA-seq data. ( A ) An RNA-seq cDNA read that maps to the reference DNA in a non-linear, chiastic manner, is a hallmark of circular RNA. ( B ) Schematic representation of the Sulfolobus solfataricus tRNA Trp , which contains a 65 b intron that is cleaved in the process of tRNA maturation and becomes a stable RNA circle. ( C ) Reads derived from the region around the circularization junction of the excised tRNA Trp intron show chiastic mapping to the genome of S. solfataricus P2, exemplifying the power of RNA-seq in circular RNA discovery. Multiple different reads spanning the circularization junction confirm that the observed circular junction is not an amplification artifact. Numbers denote position of the S. solfataricus P2 genome.
Figure Legend Snippet: Identification of circular RNA products in RNA-seq data. ( A ) An RNA-seq cDNA read that maps to the reference DNA in a non-linear, chiastic manner, is a hallmark of circular RNA. ( B ) Schematic representation of the Sulfolobus solfataricus tRNA Trp , which contains a 65 b intron that is cleaved in the process of tRNA maturation and becomes a stable RNA circle. ( C ) Reads derived from the region around the circularization junction of the excised tRNA Trp intron show chiastic mapping to the genome of S. solfataricus P2, exemplifying the power of RNA-seq in circular RNA discovery. Multiple different reads spanning the circularization junction confirm that the observed circular junction is not an amplification artifact. Numbers denote position of the S. solfataricus P2 genome.

Techniques Used: RNA Sequencing Assay, Derivative Assay, Amplification

Experimental verification of cRNAs by RT-PCR. ( A ) Left, RT–PCR results of amplification with outward-directed primers, designed to amplify cRNA; right, RT–PCR results of amplification with inward-directed primers, expected to amplify both linear and cRNA form. Single and multiple arrowheads represent single or double/triple size products, respectively. N/S, non-specific amplification (as verified by direct sequencing). RT–PCR with each primer set was performed on total RNA sample, RNase R-treated sample, and DNA sample, all extracted from S. solfataricus grown to stationary phase on organotrophic medium. ( B ) RT–PCR for verification of circular RNAs. Arrows indicate outward facing primers (top) and inward facing primers (bottom). Purple line/circle denotes the RNA template, pink line denotes expected PCR product. ( C ) Double and triple sized products can stem from multiple rounds of RT around a circular RNA template, followed by PCR amplification. Arrows mark illustrative PCR primers. ( D ) Northern blot analyses of two ncRNAs with circular forms: (M) Size marker, ( 1 ) ncRNA found in genomic location 1 275 500–1 275 567, ( 2 ) ncRNA found in genomic location 442 786–442 854. Circular forms are indicated by ‘C’, and linear forms by ‘L’.
Figure Legend Snippet: Experimental verification of cRNAs by RT-PCR. ( A ) Left, RT–PCR results of amplification with outward-directed primers, designed to amplify cRNA; right, RT–PCR results of amplification with inward-directed primers, expected to amplify both linear and cRNA form. Single and multiple arrowheads represent single or double/triple size products, respectively. N/S, non-specific amplification (as verified by direct sequencing). RT–PCR with each primer set was performed on total RNA sample, RNase R-treated sample, and DNA sample, all extracted from S. solfataricus grown to stationary phase on organotrophic medium. ( B ) RT–PCR for verification of circular RNAs. Arrows indicate outward facing primers (top) and inward facing primers (bottom). Purple line/circle denotes the RNA template, pink line denotes expected PCR product. ( C ) Double and triple sized products can stem from multiple rounds of RT around a circular RNA template, followed by PCR amplification. Arrows mark illustrative PCR primers. ( D ) Northern blot analyses of two ncRNAs with circular forms: (M) Size marker, ( 1 ) ncRNA found in genomic location 1 275 500–1 275 567, ( 2 ) ncRNA found in genomic location 442 786–442 854. Circular forms are indicated by ‘C’, and linear forms by ‘L’.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Polymerase Chain Reaction, Northern Blot, Marker

21) Product Images from "An improved method for isolation of RNA from bone"

Article Title: An improved method for isolation of RNA from bone

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-12-5

Extracting High Quality RNA from Bone in a Single Step . (A) Isolated liver RNA was incubated with RNase free beads (lane 1) or untreated beads (lane 2) provided by the manufacturer (Next Advance) for four hours before analyzing the 18S and 28S rRNA bands using agarose gel chromatography. Subsequent experiments with the Bullet Blender were carried out using untreated beads. (B) Bone RNA was homogenized in near freezing conditions using the Bullet Blender centrifuge (lanes 2-4) or a Polytron (lanes 5-6). The results are compared to bone RNA isolated using standard homogenization conditions (lane 1) and intact liver RNA that was previously isolated (control). (C) The RNA Integrity Number (RIN) for RNA homogenized in near freezing conditions was determined using the Agilent RNA 6000 Nano LabChip Kit and the Agilent 2100 Bioanalyzer. RNA with a RIN = 7 or greater is suitable for microarray analysis of gene expression. (D) The maximum RIN for the two-step approach (lane 2) is compared to the maximum RIN obtained in the one-step approach (lane 3) and each RIN is compared to the RIN of the degraded RNA sample (lane 1) shown in the agarose gel in lane 1, Figure 2B. (E) The electropherograms associated with the samples shown in (D).
Figure Legend Snippet: Extracting High Quality RNA from Bone in a Single Step . (A) Isolated liver RNA was incubated with RNase free beads (lane 1) or untreated beads (lane 2) provided by the manufacturer (Next Advance) for four hours before analyzing the 18S and 28S rRNA bands using agarose gel chromatography. Subsequent experiments with the Bullet Blender were carried out using untreated beads. (B) Bone RNA was homogenized in near freezing conditions using the Bullet Blender centrifuge (lanes 2-4) or a Polytron (lanes 5-6). The results are compared to bone RNA isolated using standard homogenization conditions (lane 1) and intact liver RNA that was previously isolated (control). (C) The RNA Integrity Number (RIN) for RNA homogenized in near freezing conditions was determined using the Agilent RNA 6000 Nano LabChip Kit and the Agilent 2100 Bioanalyzer. RNA with a RIN = 7 or greater is suitable for microarray analysis of gene expression. (D) The maximum RIN for the two-step approach (lane 2) is compared to the maximum RIN obtained in the one-step approach (lane 3) and each RIN is compared to the RIN of the degraded RNA sample (lane 1) shown in the agarose gel in lane 1, Figure 2B. (E) The electropherograms associated with the samples shown in (D).

Techniques Used: Isolation, Incubation, Agarose Gel Electrophoresis, Chromatography, Homogenization, Microarray, Expressing

The Isolated Bone RNA Supports Gene Expression Analysis using Real-time qRT-PCR . One μg of the isolated RNA was reverse transcribed using Multiscribe Reverse Transcriptase (Applied Biosystems) with random primers at 37°C for 2 hours. Gene expression of the bone specific transcription factor Runt-related transcription factor 2 (Cbfa1/Runx2) and the adipocyte-specific transcription factor Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) was analyzed via real-time PCR performed with TaqMan chemistry using the 7900 Real-time PCR system and universal cycling conditions. The Runx2 and PPARγ TaqMan primer-probe pairs were obtained from Applied Biosystems. (A) The amplification plot and (B) standard curve generated for Runx2. (C) Comparison of the gene expression of PPARγ and Runx2 from bone RNA isolated from the one-step approach versus multi-step methods. The bone was obtained from an animal fed a high fat diet for 4 months.
Figure Legend Snippet: The Isolated Bone RNA Supports Gene Expression Analysis using Real-time qRT-PCR . One μg of the isolated RNA was reverse transcribed using Multiscribe Reverse Transcriptase (Applied Biosystems) with random primers at 37°C for 2 hours. Gene expression of the bone specific transcription factor Runt-related transcription factor 2 (Cbfa1/Runx2) and the adipocyte-specific transcription factor Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) was analyzed via real-time PCR performed with TaqMan chemistry using the 7900 Real-time PCR system and universal cycling conditions. The Runx2 and PPARγ TaqMan primer-probe pairs were obtained from Applied Biosystems. (A) The amplification plot and (B) standard curve generated for Runx2. (C) Comparison of the gene expression of PPARγ and Runx2 from bone RNA isolated from the one-step approach versus multi-step methods. The bone was obtained from an animal fed a high fat diet for 4 months.

Techniques Used: Isolation, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Amplification, Generated

22) Product Images from "Estimating RNA-quality using GeneChip microarrays"

Article Title: Estimating RNA-quality using GeneChip microarrays

Journal: BMC Genomics

doi: 10.1186/1471-2164-13-186

Comparison of microarray degradation measures (d k and 5′/3′-ratio of the hybridization controls) with the RNA integrity number (RIN, panel a and c) and with the mean length of the transcripts (panel b) obtained in the ratQC experiment. [ 10 ] The d k parameter split into two branches for the two sample treatments when plotted as a function of RIN whereas the d k data virtually merge into one branch if plotted as a function of transcript length. Panel d correlates the d k and the 5′/3′- intensity ratio of the control probes in logarithmic scale. The vertical and horizontal orange lines indicate the respective quality thresholds. Good RNA-quality probes are found in direction of the arrows
Figure Legend Snippet: Comparison of microarray degradation measures (d k and 5′/3′-ratio of the hybridization controls) with the RNA integrity number (RIN, panel a and c) and with the mean length of the transcripts (panel b) obtained in the ratQC experiment. [ 10 ] The d k parameter split into two branches for the two sample treatments when plotted as a function of RIN whereas the d k data virtually merge into one branch if plotted as a function of transcript length. Panel d correlates the d k and the 5′/3′- intensity ratio of the control probes in logarithmic scale. The vertical and horizontal orange lines indicate the respective quality thresholds. Good RNA-quality probes are found in direction of the arrows

Techniques Used: Microarray, Hybridization

23) Product Images from "Label-Free Quantification of MicroRNAs Using Ligase-Assisted Sandwich Hybridization on a DNA Microarray"

Article Title: Label-Free Quantification of MicroRNAs Using Ligase-Assisted Sandwich Hybridization on a DNA Microarray

Journal: PLoS ONE

doi: 10.1371/journal.pone.0090920

Quantification of miRNAs in total RNA samples derived from two types of human blood. The amounts of miRNAs in 1 µg of total RNA were estimated by qRT-PCR or LASH assay. The expression profiles of the two blood samples were reproducible between the two methods. Data represent the mean ± S.E. (n = 3).
Figure Legend Snippet: Quantification of miRNAs in total RNA samples derived from two types of human blood. The amounts of miRNAs in 1 µg of total RNA were estimated by qRT-PCR or LASH assay. The expression profiles of the two blood samples were reproducible between the two methods. Data represent the mean ± S.E. (n = 3).

Techniques Used: Derivative Assay, Quantitative RT-PCR, Expressing

24) Product Images from "Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip)"

Article Title: Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip)

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq024

Real-time qRT-PCR analysis of TS and p53 mRNA expression from total RNA isolated from control and 5-FU-treated HCT116 cells ( n = 3, P
Figure Legend Snippet: Real-time qRT-PCR analysis of TS and p53 mRNA expression from total RNA isolated from control and 5-FU-treated HCT116 cells ( n = 3, P

Techniques Used: Quantitative RT-PCR, Expressing, Isolation

25) Product Images from "Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing"

Article Title: Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing

Journal: BMC Medical Genomics

doi: 10.1186/s12920-015-0109-x

Illustration of study design and samples. Human biological samples ( N = 45) were included in the present study. a Peripheral blood from a single individual was split into 11 aliquots (technical replicates) to test three different small RNA library purification methods: Novex TBE PAGE gel ( N = 3), Pippin Prep automated gel system (PPS) ( N = 4), and AMPure XP beads (( N = 3). Sample C1 (control-human brain) ( N = 1), sample AC (control-no purification method) ( N = 1). b Peripheral blood from a single individual was split into 5 aliquots (technical replicates) to test optimal amounts of RNA input: (1 μg), (0.5 μg), (0.25 μg), (0.1 μg), and (0.05 μg). All libraries were purified using the PPS system. c Peripheral blood samples from 15 healthy volunteers (biological replicates) to test the effects of RNA integrity. Samples were split into 5 groups ( N = 3) with average RIN values of 9, 7, 5.4, 2.2 and 0. All libraries were purified using AMPure XP beads. d Peripheral blood samples from 12 healthy volunteers (biological replicates) to test effects of sequencing coverage. Samples sequenced on both a HiSeq2500 ( N = 12) and MiSeq ( N = 12) Illumina sequencers. All libraries were purified using AMPure XP beads. e Human whole-blood ( N = 4), brain ( N = 4), heart ( N = 4) and liver ( N = 4) tissues to test expression and tissue specificity of small ncRNAs. All libraries were purified using AMPure XP beads
Figure Legend Snippet: Illustration of study design and samples. Human biological samples ( N = 45) were included in the present study. a Peripheral blood from a single individual was split into 11 aliquots (technical replicates) to test three different small RNA library purification methods: Novex TBE PAGE gel ( N = 3), Pippin Prep automated gel system (PPS) ( N = 4), and AMPure XP beads (( N = 3). Sample C1 (control-human brain) ( N = 1), sample AC (control-no purification method) ( N = 1). b Peripheral blood from a single individual was split into 5 aliquots (technical replicates) to test optimal amounts of RNA input: (1 μg), (0.5 μg), (0.25 μg), (0.1 μg), and (0.05 μg). All libraries were purified using the PPS system. c Peripheral blood samples from 15 healthy volunteers (biological replicates) to test the effects of RNA integrity. Samples were split into 5 groups ( N = 3) with average RIN values of 9, 7, 5.4, 2.2 and 0. All libraries were purified using AMPure XP beads. d Peripheral blood samples from 12 healthy volunteers (biological replicates) to test effects of sequencing coverage. Samples sequenced on both a HiSeq2500 ( N = 12) and MiSeq ( N = 12) Illumina sequencers. All libraries were purified using AMPure XP beads. e Human whole-blood ( N = 4), brain ( N = 4), heart ( N = 4) and liver ( N = 4) tissues to test expression and tissue specificity of small ncRNAs. All libraries were purified using AMPure XP beads

Techniques Used: Purification, Polyacrylamide Gel Electrophoresis, Sequencing, Expressing

26) Product Images from "Controlled electromechanical cell stimulation on-a-chip"

Article Title: Controlled electromechanical cell stimulation on-a-chip

Journal: Scientific Reports

doi: 10.1038/srep11800

Gene expression analysis. A ) Assessment of RNA qualities and concentrations using the Agilent Bioanalyzer RNA 6000 Pico Kit. RNA concentrations showed in the electropherograms varied from ~800 pg/μl to ~2 ng/μl, with an integrity number of 8.9 ± 0.1 representing an high quality RNA. ( B ) Agilent Bioanalyzer gel image of total RNA. ( C ) Histogram showing fold-changes in the expression of the selected genes following mechanical stimulation (7% strain), electrical stimulations (5 V/cm) and electromechanical stimulation (strain 7%, electrical signal 5 V/cm) compared to a control configuration without stimulation. GAPDH was chosen as housekeeping gene. Data are expressed as means ± SD.
Figure Legend Snippet: Gene expression analysis. A ) Assessment of RNA qualities and concentrations using the Agilent Bioanalyzer RNA 6000 Pico Kit. RNA concentrations showed in the electropherograms varied from ~800 pg/μl to ~2 ng/μl, with an integrity number of 8.9 ± 0.1 representing an high quality RNA. ( B ) Agilent Bioanalyzer gel image of total RNA. ( C ) Histogram showing fold-changes in the expression of the selected genes following mechanical stimulation (7% strain), electrical stimulations (5 V/cm) and electromechanical stimulation (strain 7%, electrical signal 5 V/cm) compared to a control configuration without stimulation. GAPDH was chosen as housekeeping gene. Data are expressed as means ± SD.

Techniques Used: Expressing

27) Product Images from "Type I Interferon Regulates the Expression of Long Non-Coding RNAs"

Article Title: Type I Interferon Regulates the Expression of Long Non-Coding RNAs

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2014.00548

Microarray analysis of samples treated with IFN . HuH7 cells were treated for 72 h with 0 or 10,000 units/ml of IFNα2 in three independent experiments. RNA isolated from these cells was hybridized to an Agilent array that interrogates the expression of 27958 Entrez genes and 7419 lncRNAs. Heat map clustering of all genes with B > 7 (A) , B > 1.5 (B) , or the curated probes described as lincRNAs with B > 1.5 (D) is shown. Ingenuity analysis of the set described in B is also shown (C) . The color scale is indicated for each heat map and uses log 2 units. Upregulated probes are shown in red. Downregulated probes are shown in green.
Figure Legend Snippet: Microarray analysis of samples treated with IFN . HuH7 cells were treated for 72 h with 0 or 10,000 units/ml of IFNα2 in three independent experiments. RNA isolated from these cells was hybridized to an Agilent array that interrogates the expression of 27958 Entrez genes and 7419 lncRNAs. Heat map clustering of all genes with B > 7 (A) , B > 1.5 (B) , or the curated probes described as lincRNAs with B > 1.5 (D) is shown. Ingenuity analysis of the set described in B is also shown (C) . The color scale is indicated for each heat map and uses log 2 units. Upregulated probes are shown in red. Downregulated probes are shown in green.

Techniques Used: Microarray, Isolation, Expressing

28) Product Images from "Ensuring good quality rna for quantitative real-time pcr isolated from renal proximal tubular cells using laser capture microdissection"

Article Title: Ensuring good quality rna for quantitative real-time pcr isolated from renal proximal tubular cells using laser capture microdissection

Journal: BMC Research Notes

doi: 10.1186/1756-0500-7-62

Total RNA concentrations from different number of renal cells. Total RNA concentrations were measured using Agilent 2100 Bioanalyzer with Agilent RNA 6000 Pico Kit. It was observed that 3000 renal cells yielded a total RNA concentration too low to be detected while 8000 and 18000 renal cells produced similar total RNA yields.
Figure Legend Snippet: Total RNA concentrations from different number of renal cells. Total RNA concentrations were measured using Agilent 2100 Bioanalyzer with Agilent RNA 6000 Pico Kit. It was observed that 3000 renal cells yielded a total RNA concentration too low to be detected while 8000 and 18000 renal cells produced similar total RNA yields.

Techniques Used: Concentration Assay, Produced

29) Product Images from "Microarray Analysis of Rice d1 (RGA1) Mutant Reveals the Potential Role of G-Protein Alpha Subunit in Regulating Multiple Abiotic Stresses Such as Drought, Salinity, Heat, and Cold"

Article Title: Microarray Analysis of Rice d1 (RGA1) Mutant Reveals the Potential Role of G-Protein Alpha Subunit in Regulating Multiple Abiotic Stresses Such as Drought, Salinity, Heat, and Cold

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2016.00011

qRT-PCR validation of RGA1-regulated genes identified as common to various abiotic stresses . Out of the 1498 RGA1-regulated genes identified as common to multiple abiotic stresses on the microarray, 12 of the most up/down-regulated genes were validated by qRT-PCR. Their fold change data are shown based on averages of two biological replicates and two technical replicates of total RNA, along with microarray results. The left panel in red shows the up-regulated genes and the right panel in green shows the down-regulated genes.
Figure Legend Snippet: qRT-PCR validation of RGA1-regulated genes identified as common to various abiotic stresses . Out of the 1498 RGA1-regulated genes identified as common to multiple abiotic stresses on the microarray, 12 of the most up/down-regulated genes were validated by qRT-PCR. Their fold change data are shown based on averages of two biological replicates and two technical replicates of total RNA, along with microarray results. The left panel in red shows the up-regulated genes and the right panel in green shows the down-regulated genes.

Techniques Used: Quantitative RT-PCR, Microarray

30) Product Images from "Mutation of EMG1 causing Bowen–Conradi syndrome results in reduced cell proliferation rates concomitant with G2/M arrest and 18S rRNA processing delay"

Article Title: Mutation of EMG1 causing Bowen–Conradi syndrome results in reduced cell proliferation rates concomitant with G2/M arrest and 18S rRNA processing delay

Journal: BBA Clinical

doi: 10.1016/j.bbacli.2014.05.002

Ribosomal RNA processing is delayed in BCS patient cells. (A) In fibroblasts, nascent rRNA was metabolically labeled using (methyl- 3 H) methionine for 30 min, and RNA was isolated at twenty minute intervals to follow the processing rate of the large 45S rRNA precursor to the mature 18S (arrowhead) and 28S species (arrow). Equal counts were separated on a 0.7% agarose gel, and transferred to a positively-charged nylon membrane. The membrane was exposed to a phosphor storage screen, which was scanned using a phosphorimager. The precursor 45S rRNA, the 28S and 18S mature species, and the intermediates are indicated on the left side of the diagram. A representative image of three independent experiments is shown. (B, C) The intensity of each band in (A) was quantified using Image Lab software. The graphs indicate the mean density in arbitrary units of the 18S (B) and 28S rRNA (C) bands at each time point, and the error bars represent standard deviation. For this experiment, the average of three control cell lines and two BCS cell lines are shown. The 18S rRNA level was significantly reduced in BCS cells at the 20 minute time point (*p = 0.0287). (D) A representative image of an rRNA processing experiment in lymphoblasts. The experiment was performed essentially as in (A), except the rRNA was labeled with 32 P i for a twenty minute pulse. Following gel electrophoresis to separate the RNA species, the gel was dried and directly exposed to a phosphor storage screen. (E) and (F) show the intensity of each band. The 18S rRNA was significantly reduced in BCS cells at the 40 minute time point (*p = 0.0292).
Figure Legend Snippet: Ribosomal RNA processing is delayed in BCS patient cells. (A) In fibroblasts, nascent rRNA was metabolically labeled using (methyl- 3 H) methionine for 30 min, and RNA was isolated at twenty minute intervals to follow the processing rate of the large 45S rRNA precursor to the mature 18S (arrowhead) and 28S species (arrow). Equal counts were separated on a 0.7% agarose gel, and transferred to a positively-charged nylon membrane. The membrane was exposed to a phosphor storage screen, which was scanned using a phosphorimager. The precursor 45S rRNA, the 28S and 18S mature species, and the intermediates are indicated on the left side of the diagram. A representative image of three independent experiments is shown. (B, C) The intensity of each band in (A) was quantified using Image Lab software. The graphs indicate the mean density in arbitrary units of the 18S (B) and 28S rRNA (C) bands at each time point, and the error bars represent standard deviation. For this experiment, the average of three control cell lines and two BCS cell lines are shown. The 18S rRNA level was significantly reduced in BCS cells at the 20 minute time point (*p = 0.0287). (D) A representative image of an rRNA processing experiment in lymphoblasts. The experiment was performed essentially as in (A), except the rRNA was labeled with 32 P i for a twenty minute pulse. Following gel electrophoresis to separate the RNA species, the gel was dried and directly exposed to a phosphor storage screen. (E) and (F) show the intensity of each band. The 18S rRNA was significantly reduced in BCS cells at the 40 minute time point (*p = 0.0292).

Techniques Used: Metabolic Labelling, Labeling, Isolation, Agarose Gel Electrophoresis, Software, Standard Deviation, Nucleic Acid Electrophoresis

Ribosomal RNA levels at steady-state are normal in BCS patient cells. (A, B) Total RNA was isolated from unaffected control and BCS-affected lymphoblasts (A) and fibroblasts (B), and separated by capillary chromatography using the 6000 RNA Nano kit in an Agilent Bioanalyzer. The resulting electropherograms show the 18S and 28S peaks, as well as a smaller peak which encompasses the 5S, 5.8S, and tRNA. (C, D) 28S/18S ratios for lymphoblasts (C) and fibroblasts (D). The area of each peak was calculated by the Agilent software, and the ratios of 28S to 18S rRNA were calculated. The mean of three individual experiments, performed in triplicate, and SEM are shown. No significant difference was found between the control and BCS patient cells.
Figure Legend Snippet: Ribosomal RNA levels at steady-state are normal in BCS patient cells. (A, B) Total RNA was isolated from unaffected control and BCS-affected lymphoblasts (A) and fibroblasts (B), and separated by capillary chromatography using the 6000 RNA Nano kit in an Agilent Bioanalyzer. The resulting electropherograms show the 18S and 28S peaks, as well as a smaller peak which encompasses the 5S, 5.8S, and tRNA. (C, D) 28S/18S ratios for lymphoblasts (C) and fibroblasts (D). The area of each peak was calculated by the Agilent software, and the ratios of 28S to 18S rRNA were calculated. The mean of three individual experiments, performed in triplicate, and SEM are shown. No significant difference was found between the control and BCS patient cells.

Techniques Used: Isolation, Chromatography, Software

31) Product Images from "Long Term Storage of Dry versus Frozen RNA for Next Generation Molecular Studies"

Article Title: Long Term Storage of Dry versus Frozen RNA for Next Generation Molecular Studies

Journal: PLoS ONE

doi: 10.1371/journal.pone.0111827

RNA Integrity Number analysis of stored RNA. ( A ) RIN Electrophoretic Analysis of RNA integrity from two representative RNA samples which were desiccated and stored at room temperature (D) or Frozen at −80°C (F) for 12 months. Desiccated samples are shown on the left and frozen samples are shown on the right. The two main peaks in each electropherogram are representative of 18S and 28S ribosomal RNA fragments. ( B ) The average RIN values with standard error of the mean from all RNA samples measured after the given number of months in storage in a desiccated (D) or frozen (F) state.
Figure Legend Snippet: RNA Integrity Number analysis of stored RNA. ( A ) RIN Electrophoretic Analysis of RNA integrity from two representative RNA samples which were desiccated and stored at room temperature (D) or Frozen at −80°C (F) for 12 months. Desiccated samples are shown on the left and frozen samples are shown on the right. The two main peaks in each electropherogram are representative of 18S and 28S ribosomal RNA fragments. ( B ) The average RIN values with standard error of the mean from all RNA samples measured after the given number of months in storage in a desiccated (D) or frozen (F) state.

Techniques Used:

32) Product Images from "Inhibition of miR-21 in glioma cells using catalytic nucleic acids"

Article Title: Inhibition of miR-21 in glioma cells using catalytic nucleic acids

Journal: Scientific Reports

doi: 10.1038/srep24516

The effect of anti-miR-21 agents on the endogenous miR-21, miR-21 precursors and PTEN pools in T98G cells. T98G cells were transfected using Lipofectamine 2000 Transfection Reagent with: anti-miR-21 ribozymes (miR21rz1, miR21rz2, miR21rz3, miR21rz1_mut, 100 nM each), anti-miR-21 DNAzymes (miR21dz1, miR21dz2 and miR21dz3, miR21dz4, miR21dz5, 100 nM each) and antisense anti-miR-21 antagomir with LNA modifications (LNA, 50 nM). Non-transfected T98G cells treated only with Lipofectamine 2000 served as negative control. 24 h post-transfection cells were harvested and total RNA was isolated. RNA samples were treated with DNase I and assessed in terms of quantity and quality. They were polyadenylated and reverse-transcribed. Relative expression of miR-21 and pre-miR-21 was quantified using miR-21 forward primer and Universal Reverse Primer in qPCR using LightCycler 480 System and normalized using 18S rRNA as reference and E-Method for calculation of relative expression of miR-21 and pre-miR-21. Additionally, 24 h post-transfection cells were harvested and total protein lysate was prepared. Samples were assessed in terms of quantity and quality. Relative expression of PTEN was quantified in Western Blot and normalized using GAPDH as reference.
Figure Legend Snippet: The effect of anti-miR-21 agents on the endogenous miR-21, miR-21 precursors and PTEN pools in T98G cells. T98G cells were transfected using Lipofectamine 2000 Transfection Reagent with: anti-miR-21 ribozymes (miR21rz1, miR21rz2, miR21rz3, miR21rz1_mut, 100 nM each), anti-miR-21 DNAzymes (miR21dz1, miR21dz2 and miR21dz3, miR21dz4, miR21dz5, 100 nM each) and antisense anti-miR-21 antagomir with LNA modifications (LNA, 50 nM). Non-transfected T98G cells treated only with Lipofectamine 2000 served as negative control. 24 h post-transfection cells were harvested and total RNA was isolated. RNA samples were treated with DNase I and assessed in terms of quantity and quality. They were polyadenylated and reverse-transcribed. Relative expression of miR-21 and pre-miR-21 was quantified using miR-21 forward primer and Universal Reverse Primer in qPCR using LightCycler 480 System and normalized using 18S rRNA as reference and E-Method for calculation of relative expression of miR-21 and pre-miR-21. Additionally, 24 h post-transfection cells were harvested and total protein lysate was prepared. Samples were assessed in terms of quantity and quality. Relative expression of PTEN was quantified in Western Blot and normalized using GAPDH as reference.

Techniques Used: Transfection, Negative Control, Isolation, Expressing, Real-time Polymerase Chain Reaction, Western Blot

33) Product Images from "RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells"

Article Title: RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0141070

Overview of experimental structure. Human bone marrow-derived multipotent stromal cell (BM-MSC) lines were cultivated and expanded until passage 3 or 4. Following the RNA was isolated and the cDNA preparation was performed by standard reverse transcription and by Whole Transcriptome Amplification (WTA). The same RNA of the respective human BM-MSC line was applied for standard reverse transcription and WTA to compare RT- and qPCR results of the non-amplified and amplified cDNA samples. To exclude RNA degradation, the RNA integrity number (RIN) was determined by Agilent Bioanalyzer and only RNA samples with RIN values ≥ 9.8 were applied. Additionally, various amounts of RNA and cDNA were tested as starting material for WTA or for RT-PCR analysis.
Figure Legend Snippet: Overview of experimental structure. Human bone marrow-derived multipotent stromal cell (BM-MSC) lines were cultivated and expanded until passage 3 or 4. Following the RNA was isolated and the cDNA preparation was performed by standard reverse transcription and by Whole Transcriptome Amplification (WTA). The same RNA of the respective human BM-MSC line was applied for standard reverse transcription and WTA to compare RT- and qPCR results of the non-amplified and amplified cDNA samples. To exclude RNA degradation, the RNA integrity number (RIN) was determined by Agilent Bioanalyzer and only RNA samples with RIN values ≥ 9.8 were applied. Additionally, various amounts of RNA and cDNA were tested as starting material for WTA or for RT-PCR analysis.

Techniques Used: Derivative Assay, Isolation, Amplification, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

34) Product Images from "RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells"

Article Title: RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0141070

Overview of experimental structure. Human bone marrow-derived multipotent stromal cell (BM-MSC) lines were cultivated and expanded until passage 3 or 4. Following the RNA was isolated and the cDNA preparation was performed by standard reverse transcription and by Whole Transcriptome Amplification (WTA). The same RNA of the respective human BM-MSC line was applied for standard reverse transcription and WTA to compare RT- and qPCR results of the non-amplified and amplified cDNA samples. To exclude RNA degradation, the RNA integrity number (RIN) was determined by Agilent Bioanalyzer and only RNA samples with RIN values ≥ 9.8 were applied. Additionally, various amounts of RNA and cDNA were tested as starting material for WTA or for RT-PCR analysis.
Figure Legend Snippet: Overview of experimental structure. Human bone marrow-derived multipotent stromal cell (BM-MSC) lines were cultivated and expanded until passage 3 or 4. Following the RNA was isolated and the cDNA preparation was performed by standard reverse transcription and by Whole Transcriptome Amplification (WTA). The same RNA of the respective human BM-MSC line was applied for standard reverse transcription and WTA to compare RT- and qPCR results of the non-amplified and amplified cDNA samples. To exclude RNA degradation, the RNA integrity number (RIN) was determined by Agilent Bioanalyzer and only RNA samples with RIN values ≥ 9.8 were applied. Additionally, various amounts of RNA and cDNA were tested as starting material for WTA or for RT-PCR analysis.

Techniques Used: Derivative Assay, Isolation, Amplification, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

35) Product Images from "Systematic interactome mapping of acute lymphoblastic leukemia cancer gene products reveals EXT-1 tumor suppressor as a Notch1 and FBWX7 common interactor"

Article Title: Systematic interactome mapping of acute lymphoblastic leukemia cancer gene products reveals EXT-1 tumor suppressor as a Notch1 and FBWX7 common interactor

Journal: BMC Cancer

doi: 10.1186/s12885-016-2374-2

Genes coregulated by EXT1 and FBXW7. a HeLa Notch∆E-eGFP were treated siRNA for EXT1, FBXW7 or a control siRNA. Relative mRNA expression levels of EXT1 and FBXW7 were then analyzed by qPCR and RNA samples subjected to high throughput Illumina sequencing (RNA-seq). Ven diagrams represent a comparison between deregulated genes following knock down of EXT1 or FBXW7. b Relative mRNA expression levels of EXT1 and FBXW7 analyzed by qPCR. c Gene ontology enrichment analysis of common deregulated genes. GO-terms and node sizes are proportional to the number of genes implicated in the same GO term
Figure Legend Snippet: Genes coregulated by EXT1 and FBXW7. a HeLa Notch∆E-eGFP were treated siRNA for EXT1, FBXW7 or a control siRNA. Relative mRNA expression levels of EXT1 and FBXW7 were then analyzed by qPCR and RNA samples subjected to high throughput Illumina sequencing (RNA-seq). Ven diagrams represent a comparison between deregulated genes following knock down of EXT1 or FBXW7. b Relative mRNA expression levels of EXT1 and FBXW7 analyzed by qPCR. c Gene ontology enrichment analysis of common deregulated genes. GO-terms and node sizes are proportional to the number of genes implicated in the same GO term

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, High Throughput Screening Assay, Sequencing, RNA Sequencing Assay

36) Product Images from "Exosomes from bulk and stem cells from human prostate cancer have a differential microRNA content that contributes cooperatively over local and pre-metastatic niche"

Article Title: Exosomes from bulk and stem cells from human prostate cancer have a differential microRNA content that contributes cooperatively over local and pre-metastatic niche

Journal: Oncotarget

doi: 10.18632/oncotarget.6540

RNA profiles from exosomes from bulk cells (left) and CSCs (right) exosomes obtained by Agilent 2100 Bioanalyzer The electropherograms shows the size in distribution of the nucleotides (nt) and fluorescence intensity (FU) of Total RNA by Agilent RNA 6000 Pico Kit (A) and small RNAs after enrichment by the Agilent Small RNA Kit (B) All the electropherograms correspond to the sample of the same patient. The inserts show a representative image from the cultures under study.
Figure Legend Snippet: RNA profiles from exosomes from bulk cells (left) and CSCs (right) exosomes obtained by Agilent 2100 Bioanalyzer The electropherograms shows the size in distribution of the nucleotides (nt) and fluorescence intensity (FU) of Total RNA by Agilent RNA 6000 Pico Kit (A) and small RNAs after enrichment by the Agilent Small RNA Kit (B) All the electropherograms correspond to the sample of the same patient. The inserts show a representative image from the cultures under study.

Techniques Used: Fluorescence

37) Product Images from "High quality RNA extraction from Maqui berry for its application in next-generation sequencing"

Article Title: High quality RNA extraction from Maqui berry for its application in next-generation sequencing

Journal: SpringerPlus

doi: 10.1186/s40064-016-2906-x

Electrophoretogram of sequencing libraries. The graph shows the length distribution curves of sequencing libraries obtained with Illumina TruSeqTM RNA sample preparation kit (Low-Throughput protocol) according to manufacturer’s protocol. The length of DNA fragments was between 200 and 700 pb. Curves were generated on a 2100 bioanalyzer using DNA 1000 chip (Agilent Technologies). For each sample three biological replicates were performed
Figure Legend Snippet: Electrophoretogram of sequencing libraries. The graph shows the length distribution curves of sequencing libraries obtained with Illumina TruSeqTM RNA sample preparation kit (Low-Throughput protocol) according to manufacturer’s protocol. The length of DNA fragments was between 200 and 700 pb. Curves were generated on a 2100 bioanalyzer using DNA 1000 chip (Agilent Technologies). For each sample three biological replicates were performed

Techniques Used: Sequencing, Sample Prep, Generated, Chromatin Immunoprecipitation

Electrophoretogram of total RNA obtained with our new method. The 18S and 28S rRNA 25 regions are shown. RNA concentrations and RIN values are shown below. a green fruit. b red fruit. c blue fruit. RNA was analyzed with the Agilent RNA 6000 Nano Assay in a 2100 bioanalyzer (Agilent Technologies). Note that the output from our instrument is completely opposite from the English convention, therefore, it uses commas as an indicator of decimals, and periods to denote thousands. It is shown one result of the three RNA extraction obtained from the same tissue
Figure Legend Snippet: Electrophoretogram of total RNA obtained with our new method. The 18S and 28S rRNA 25 regions are shown. RNA concentrations and RIN values are shown below. a green fruit. b red fruit. c blue fruit. RNA was analyzed with the Agilent RNA 6000 Nano Assay in a 2100 bioanalyzer (Agilent Technologies). Note that the output from our instrument is completely opposite from the English convention, therefore, it uses commas as an indicator of decimals, and periods to denote thousands. It is shown one result of the three RNA extraction obtained from the same tissue

Techniques Used: RNA Extraction

38) Product Images from "A Conserved MicroRNA Regulatory Circuit Is Differentially Controlled during Limb/Appendage Regeneration"

Article Title: A Conserved MicroRNA Regulatory Circuit Is Differentially Controlled during Limb/Appendage Regeneration

Journal: PLoS ONE

doi: 10.1371/journal.pone.0157106

miRNA expression profiling by small RNA sequencing. (a) Overview of shared and unique miRNAs detected in regenerating limb/appendages following blastema formation in zebrafish caudal fin, bichir pectoral fins and axolotl forelimbs. (b) Gene expression patterns of differentially expressed zebrafish miRNAs shown as a heat map of log 2 -transformed read counts per million, in triplicate. (c) Relationship among fold-change and average level of expression for each miRNA in each taxa with miR-21 and miR-133. (dpa = days post-amputation).
Figure Legend Snippet: miRNA expression profiling by small RNA sequencing. (a) Overview of shared and unique miRNAs detected in regenerating limb/appendages following blastema formation in zebrafish caudal fin, bichir pectoral fins and axolotl forelimbs. (b) Gene expression patterns of differentially expressed zebrafish miRNAs shown as a heat map of log 2 -transformed read counts per million, in triplicate. (c) Relationship among fold-change and average level of expression for each miRNA in each taxa with miR-21 and miR-133. (dpa = days post-amputation).

Techniques Used: Expressing, RNA Sequencing Assay, Transformation Assay

Analyses of conserved sequence tags reveal processed tRNA fragments and isomiRs. (a) Analysis workflow used to identify conserved sequence tags. Two-hundred-forty sequence tags expressed in all zebrafish samples were also expressed in all bichir and axolotl samples. 232 sequence tags were isomiRs including 4 that had substitutions in the seed sequence. Of the eight sequence tags were not derived from miRNAs, 4 mapped to ribosomal RNAs (rRNAs) and 4 were processed tRNA fragments. (b) The 4 processed tRNA fragments expressed in all zebrafish, bichir and axolotl samples. We found these processed tRNAs to also be small RNA sequence data from in chicken, mouse and human tissues [ 78 ].
Figure Legend Snippet: Analyses of conserved sequence tags reveal processed tRNA fragments and isomiRs. (a) Analysis workflow used to identify conserved sequence tags. Two-hundred-forty sequence tags expressed in all zebrafish samples were also expressed in all bichir and axolotl samples. 232 sequence tags were isomiRs including 4 that had substitutions in the seed sequence. Of the eight sequence tags were not derived from miRNAs, 4 mapped to ribosomal RNAs (rRNAs) and 4 were processed tRNA fragments. (b) The 4 processed tRNA fragments expressed in all zebrafish, bichir and axolotl samples. We found these processed tRNAs to also be small RNA sequence data from in chicken, mouse and human tissues [ 78 ].

Techniques Used: Sequencing, Derivative Assay

39) Product Images from "High CXCR3 expression in synovial mast cells associated with CXCL9 and CXCL10 expression in inflammatory synovial tissues of patients with rheumatoid arthritis"

Article Title: High CXCR3 expression in synovial mast cells associated with CXCL9 and CXCL10 expression in inflammatory synovial tissues of patients with rheumatoid arthritis

Journal: Arthritis Research & Therapy

doi:

Analysis of IL-6 mRNA levels within synovial tissue from rheumatoid arthritis (RA) as compared with that from osteoarthritis (OA) patients. Upper panels: quality control of total RNA preparations. Aliquots (300 ng) of total RNA extracted from synovial tissue from RA and OA patients were plotted on a RNA 6000 Nano-LabChip. Quality of RNA was scanned using a 2100 bioanalyzer. RNA gel electropherograms show the presence of 28S and 18S ribosomal units, indicating intact RNA of the investigated samples. Lower panels: differential IL-6 mRNA levels were determined by semiquantitative reverse transcription polymerase chain reaction (PCR). The figure shows a representative analysis of eight cDNA samples derived from patients with RA and of eight cDNA samples from patients with OA. cDNA samples were adjusted to equal glyceraldehyde-3-phosphate dehydrogenase (G3PDH) levels, performed by competitive PCR using an internal standard (see Materials and methods). Numbered lanes correspond to individual patients within Table 1 .
Figure Legend Snippet: Analysis of IL-6 mRNA levels within synovial tissue from rheumatoid arthritis (RA) as compared with that from osteoarthritis (OA) patients. Upper panels: quality control of total RNA preparations. Aliquots (300 ng) of total RNA extracted from synovial tissue from RA and OA patients were plotted on a RNA 6000 Nano-LabChip. Quality of RNA was scanned using a 2100 bioanalyzer. RNA gel electropherograms show the presence of 28S and 18S ribosomal units, indicating intact RNA of the investigated samples. Lower panels: differential IL-6 mRNA levels were determined by semiquantitative reverse transcription polymerase chain reaction (PCR). The figure shows a representative analysis of eight cDNA samples derived from patients with RA and of eight cDNA samples from patients with OA. cDNA samples were adjusted to equal glyceraldehyde-3-phosphate dehydrogenase (G3PDH) levels, performed by competitive PCR using an internal standard (see Materials and methods). Numbered lanes correspond to individual patients within Table 1 .

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Derivative Assay

40) Product Images from "Differential Regulation of Breast Cancer-Associated Genes by Progesterone Receptor Isoforms PRA and PRB in a New Bi-Inducible Breast Cancer Cell Line"

Article Title: Differential Regulation of Breast Cancer-Associated Genes by Progesterone Receptor Isoforms PRA and PRB in a New Bi-Inducible Breast Cancer Cell Line

Journal: PLoS ONE

doi: 10.1371/journal.pone.0045993

Gene expression profiles following PR isoform conditional expression and hormonal treatment. The iPRAB cells were treated for 24 h by RSL1 (0.25 µM) and/or Dox (1 µM) to induce expression of either PRA (A) or PRB (B) or PRA plus PRB (AB) expression and then incubated with vehicle (−) or 10 −8 M progesterone (+) for 6 h. Control cells were not treated by inducers (O). PR isoforms expression was analyzed by western-blot (upper left inset). PRA/PRB ratio value corresponding to each condition was estimated according to ligand binding assays and western blot quantification as described in Material and Methods (A: 45; B: 0.04; AB: 1). The corresponding total RNAs (O−, O+, A−, A+, B−, B+, AB−, AB+) were extracted and gene expression profiling was done using Agilent 44K-oligonucleotide microarrays as described in Materials and Methods . Venn diagrams showing the total number of genes regulated for each subset and clustering analyses (heat maps) of differentially expressed genes in each experimental condition were performed as described in Materials and Methods . Each conditional expression as compared to the reference RNA is indicated on the top of heat maps. The gene clusters corresponding to Venn diagram areas are indicated on the left side of heat maps (−: ligand-independent; + ligand-dependent; ±: mixed regulation; : intersection). As indicated in the lower right panel, color intensities reflect the log 2 (ratio) values obtained for down-regulated (green) and up-regulated (red) genes, and were saturated at log 2 (ratio) of ±2 corresponding to FC ±4.
Figure Legend Snippet: Gene expression profiles following PR isoform conditional expression and hormonal treatment. The iPRAB cells were treated for 24 h by RSL1 (0.25 µM) and/or Dox (1 µM) to induce expression of either PRA (A) or PRB (B) or PRA plus PRB (AB) expression and then incubated with vehicle (−) or 10 −8 M progesterone (+) for 6 h. Control cells were not treated by inducers (O). PR isoforms expression was analyzed by western-blot (upper left inset). PRA/PRB ratio value corresponding to each condition was estimated according to ligand binding assays and western blot quantification as described in Material and Methods (A: 45; B: 0.04; AB: 1). The corresponding total RNAs (O−, O+, A−, A+, B−, B+, AB−, AB+) were extracted and gene expression profiling was done using Agilent 44K-oligonucleotide microarrays as described in Materials and Methods . Venn diagrams showing the total number of genes regulated for each subset and clustering analyses (heat maps) of differentially expressed genes in each experimental condition were performed as described in Materials and Methods . Each conditional expression as compared to the reference RNA is indicated on the top of heat maps. The gene clusters corresponding to Venn diagram areas are indicated on the left side of heat maps (−: ligand-independent; + ligand-dependent; ±: mixed regulation; : intersection). As indicated in the lower right panel, color intensities reflect the log 2 (ratio) values obtained for down-regulated (green) and up-regulated (red) genes, and were saturated at log 2 (ratio) of ±2 corresponding to FC ±4.

Techniques Used: Expressing, Incubation, Western Blot, Ligand Binding Assay

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Polymerase Chain Reaction:

Article Title: Laser microdissection coupled with RNA-seq analysis of porcine enterocytes infected with an obligate intracellular pathogen (Lawsonia intracellularis)
Article Snippet: .. Reverse transcription PCR In addition to the assessment of RNA quality based on the bacterial and eukaryotic ribosomal RNAs using Agilent Bioanalyzer 2100, one-step RT-PCR was applied to the RNA extracted from microdissection tissues in order to evaluate the quality of bacterial mRNA for RNA-seq analysis. .. The one-step RT-PCR (Qiagen®) was used with specific primers (Additional file : Table S1) targeting three housekeeping genes of L. intracellularis .

Isolation:

Article Title: Tumour-vasculature development via endothelial-to-mesenchymal transition after radiotherapy controls CD44v6+ cancer cell and macrophage polarization
Article Snippet: .. RNA-seq analysis Total RNA was isolated from HUVECs, and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). .. RNA-seq libraries were constructed using the SENSE mRNA-Seq Library Prep Kit (Lexogen), according to the manufacturer’s instructions, and were sequenced as 100 bp paired-end runs on the HiSeq 2000 platform (Illumina).

Laser Capture Microdissection:

Article Title: Laser microdissection coupled with RNA-seq analysis of porcine enterocytes infected with an obligate intracellular pathogen (Lawsonia intracellularis)
Article Snippet: .. Reverse transcription PCR In addition to the assessment of RNA quality based on the bacterial and eukaryotic ribosomal RNAs using Agilent Bioanalyzer 2100, one-step RT-PCR was applied to the RNA extracted from microdissection tissues in order to evaluate the quality of bacterial mRNA for RNA-seq analysis. .. The one-step RT-PCR (Qiagen®) was used with specific primers (Additional file : Table S1) targeting three housekeeping genes of L. intracellularis .

Reverse Transcription Polymerase Chain Reaction:

Article Title: Laser microdissection coupled with RNA-seq analysis of porcine enterocytes infected with an obligate intracellular pathogen (Lawsonia intracellularis)
Article Snippet: .. Reverse transcription PCR In addition to the assessment of RNA quality based on the bacterial and eukaryotic ribosomal RNAs using Agilent Bioanalyzer 2100, one-step RT-PCR was applied to the RNA extracted from microdissection tissues in order to evaluate the quality of bacterial mRNA for RNA-seq analysis. .. The one-step RT-PCR (Qiagen®) was used with specific primers (Additional file : Table S1) targeting three housekeeping genes of L. intracellularis .

Generated:

Article Title: A qualitative assessment of direct-labeled cDNA products prior to microarray analysis
Article Snippet: .. Fully degraded, partially degraded and intact RNA, generated by titrating varying amounts of RNase, were used to prepare samples for measuring Cy5 incorporation directly into the cDNA by reverse transcription of RNA by the Agilent 2100 Bioanalyzer. .. It should be noted, however, that RNase treatment can be unpredictable, since even slight deviations in temperature and timing of incubation can result in varying degrees of degradation.

Formalin-fixed Paraffin-Embedded:

Article Title: Biobanking: Objectives, Requirements, and Future Challenges—Experiences from the Munich Vascular Biobank
Article Snippet: .. Analysis of RNA Quality from FFPE Biospecimens by RIN and RNA Fragmentation RNA integrity number (RIN) was determined by Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kit (Agilent Technologies, Waldbronn, Germany) in accordance with the manufacturer’s instructions. ..

Sequencing:

Article Title: Decreasing miRNA sequencing bias using a single adapter and circularization approach
Article Snippet: .. Using RealSeq®-AC and TruSeq®, we prepared sequencing libraries from a reference sample of total RNA (Agilent) obtained from nine different human tissues and cell lines. .. We sequenced both libraries to a coverage of ten million reads and counted the number of miRNAs identified (with ten or more reads of each) by each kit at different sequencing coverages by random subsampling (Additional file : Figure S6).

Chromatin Immunoprecipitation:

Article Title: Characterization of human plasma-derived exosomal RNAs by deep sequencing
Article Snippet: .. The quantity and quality of the RNA were determined by Agilent Bioanalyzer 2100 with a Small RNA Chip for exosomal RNA, and a RNA 6000 Pico Kit for cellular RNA (Agilent Technologies, Santa Clara, CA, USA). .. Enzyme protection assayRNA isolated from the plasma exosomes was first incubated at room temperature, either with 30 units/μL of DNase I (QIAGEN) for 10 min or with 10 μg/mL RNase A for 30 min.

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    Agilent technologies rna
    Bias in miRNA detection using various <t>small-RNA</t> library preparation kits. For each kit, sequencing libraries were prepared from the miRXplore™ pool and sequenced; the sequence data were then used to calculate fold-deviations from the equimolar input and plotted as log 2 values. Densities of miRNAs within a two-fold deviation from the expected values (between vertical lines ) are considered unbiased according to [ 8 ]. Under-represented, over-represented, and accurately quantified percentages of miRNAs are shown in red font . Results for two-adapter schemes are a <t>TruSeq®</t> Small RNA, b NEBNext®, and c QIAseq. d NEXTFlex™, a scheme using two adapters with randomized sequences. e SMARTer, which uses template switching. f RealSeq®-AC, which uses a single-adapter and circularization (* p value vs other kits
    Rna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 95/100, based on 2871 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies mirnas total rna
    Identification of miR-29b-1-5p-regulated genes. a Expression profiles of messenger RNAs (mRNAs) in K562 cells transfected with miR-29b-1-5p mimic or scramble oligonucleotides then cultured with medium for 48 h were evaluated using <t>RNA-Seq</t> transcriptome analysis. Each scatter spot represents the mean raw signals of microRNA <t>(miRNA)</t> in three repeats of each treatment. b The RNA expression levels of 16 genes were significantly higher, whereas the RNA expression levels of 10 genes were significantly lower, in K562 cells after being transfected with miR-29b-1-5p mimic for 48 h. c Four genes potentially involved in the inflammatory responses were selected for further analysis. The mRNA expression levels of ANG were significantly elevated in K562 cells after coculture with interleukin (IL)-23 (20 ng/ml) for 3 days (Fig. 3 c)
    Mirnas Total Rna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mirnas total rna/product/Agilent technologies
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    Bias in miRNA detection using various small-RNA library preparation kits. For each kit, sequencing libraries were prepared from the miRXplore™ pool and sequenced; the sequence data were then used to calculate fold-deviations from the equimolar input and plotted as log 2 values. Densities of miRNAs within a two-fold deviation from the expected values (between vertical lines ) are considered unbiased according to [ 8 ]. Under-represented, over-represented, and accurately quantified percentages of miRNAs are shown in red font . Results for two-adapter schemes are a TruSeq® Small RNA, b NEBNext®, and c QIAseq. d NEXTFlex™, a scheme using two adapters with randomized sequences. e SMARTer, which uses template switching. f RealSeq®-AC, which uses a single-adapter and circularization (* p value vs other kits

    Journal: Genome Biology

    Article Title: Decreasing miRNA sequencing bias using a single adapter and circularization approach

    doi: 10.1186/s13059-018-1488-z

    Figure Lengend Snippet: Bias in miRNA detection using various small-RNA library preparation kits. For each kit, sequencing libraries were prepared from the miRXplore™ pool and sequenced; the sequence data were then used to calculate fold-deviations from the equimolar input and plotted as log 2 values. Densities of miRNAs within a two-fold deviation from the expected values (between vertical lines ) are considered unbiased according to [ 8 ]. Under-represented, over-represented, and accurately quantified percentages of miRNAs are shown in red font . Results for two-adapter schemes are a TruSeq® Small RNA, b NEBNext®, and c QIAseq. d NEXTFlex™, a scheme using two adapters with randomized sequences. e SMARTer, which uses template switching. f RealSeq®-AC, which uses a single-adapter and circularization (* p value vs other kits

    Article Snippet: Using RealSeq®-AC and TruSeq®, we prepared sequencing libraries from a reference sample of total RNA (Agilent) obtained from nine different human tissues and cell lines.

    Techniques: Sequencing

    Differential quantification of brain samples between different small RNA library preparation kits. Data obtained with either a TruSeq®, b NEBNext®, c NEXTFlex™, d QIAseq, or e SMARTer kits were compared with data obtained with RealSeq®-AC to obtain differential quantification (log 2 ) values for 276 high-confidence miRNAs. These values were plotted against the accuracy of detection of that miRNA when profiling the equimolar pool of synthetic miRNAs (Fig. 2 a–c). f–j The reverse comparison, with the differential quantification of RealSeq®-AC versus each of the other kits plotted against the accuracy of RealSeq®-AC when quantifying the synthetic pool of miRNAs. FN false negative, FP false positive. See Methods for more details

    Journal: Genome Biology

    Article Title: Decreasing miRNA sequencing bias using a single adapter and circularization approach

    doi: 10.1186/s13059-018-1488-z

    Figure Lengend Snippet: Differential quantification of brain samples between different small RNA library preparation kits. Data obtained with either a TruSeq®, b NEBNext®, c NEXTFlex™, d QIAseq, or e SMARTer kits were compared with data obtained with RealSeq®-AC to obtain differential quantification (log 2 ) values for 276 high-confidence miRNAs. These values were plotted against the accuracy of detection of that miRNA when profiling the equimolar pool of synthetic miRNAs (Fig. 2 a–c). f–j The reverse comparison, with the differential quantification of RealSeq®-AC versus each of the other kits plotted against the accuracy of RealSeq®-AC when quantifying the synthetic pool of miRNAs. FN false negative, FP false positive. See Methods for more details

    Article Snippet: Using RealSeq®-AC and TruSeq®, we prepared sequencing libraries from a reference sample of total RNA (Agilent) obtained from nine different human tissues and cell lines.

    Techniques:

    TRP53-regulated EndMT modulates the M1 and M2 populations of increased TAMs after radiotherapy. a RNA-seq data analysis of the samples represented in Fig. 4a . Venn diagram depicting differentially expressed genes in irradiated HUVECs. Reverse-regulated genes ( > 1.2-fold) between TGFβR2 knockdown+IR and Trp53 -knockdown (with and without TGFβR2 knockdown)+IR conditions (compared with IR alone) were selected (subgroup A). Only matrisome- and surface-associated genes in subset A are shown in the heatmap generated by k-means clustering (subgroup B). b GeneMANIA network analysis of subset B showing significant enrichment for the GO term ‘leukocyte migration’. Red-filled circles represent genes upregulated in the Trp53 siRNA+IR group vs. the IR-alone group. Blue-filled circles represent genes upregulated in the TGFβR2 siRNA+IR group vs. the IR-alone group. The names of leukocyte migration-related genes are indicated in red. c Immunofluorescence detection of F4/80, CD31, and Arg1, or iNOS, CD31, and F4/80 in KP tumours from WT and EC-p53KO mice, with or without irradiation (23 days after irradiation). Scale bar = 20 μm. d Quantification of F4/80 + Arg1 + and F4/80 + iNOS + cells/field (magnification, ×200) using immunofluorescence images of KP tumours from WT, EC-p53KO, and TGFβR2KD mice, with or without irradiation (23 days after irradiation). e Ratio of CD206 + in F4/80 + cells from WT and EC-p53KO tumours 7 days after irradiation, as determined by flow cytometry. f Immunofluorescence staining of F4/80 and Arg1 in WT bone marrow-derived macrophages after coculture with KP tumour-derived ECs from WT and EC-p53KO mice for 48 h after irradiation. Scale bar = 50 μm (crop, 20 μm). Flow-cytometric analysis of CD206 + , iNOS + , and EdU + cells among F4/80 + cells (magnification, ×200) is shown. For ( d – f ), error bars indicate SEM; * p

    Journal: Nature Communications

    Article Title: Tumour-vasculature development via endothelial-to-mesenchymal transition after radiotherapy controls CD44v6+ cancer cell and macrophage polarization

    doi: 10.1038/s41467-018-07470-w

    Figure Lengend Snippet: TRP53-regulated EndMT modulates the M1 and M2 populations of increased TAMs after radiotherapy. a RNA-seq data analysis of the samples represented in Fig. 4a . Venn diagram depicting differentially expressed genes in irradiated HUVECs. Reverse-regulated genes ( > 1.2-fold) between TGFβR2 knockdown+IR and Trp53 -knockdown (with and without TGFβR2 knockdown)+IR conditions (compared with IR alone) were selected (subgroup A). Only matrisome- and surface-associated genes in subset A are shown in the heatmap generated by k-means clustering (subgroup B). b GeneMANIA network analysis of subset B showing significant enrichment for the GO term ‘leukocyte migration’. Red-filled circles represent genes upregulated in the Trp53 siRNA+IR group vs. the IR-alone group. Blue-filled circles represent genes upregulated in the TGFβR2 siRNA+IR group vs. the IR-alone group. The names of leukocyte migration-related genes are indicated in red. c Immunofluorescence detection of F4/80, CD31, and Arg1, or iNOS, CD31, and F4/80 in KP tumours from WT and EC-p53KO mice, with or without irradiation (23 days after irradiation). Scale bar = 20 μm. d Quantification of F4/80 + Arg1 + and F4/80 + iNOS + cells/field (magnification, ×200) using immunofluorescence images of KP tumours from WT, EC-p53KO, and TGFβR2KD mice, with or without irradiation (23 days after irradiation). e Ratio of CD206 + in F4/80 + cells from WT and EC-p53KO tumours 7 days after irradiation, as determined by flow cytometry. f Immunofluorescence staining of F4/80 and Arg1 in WT bone marrow-derived macrophages after coculture with KP tumour-derived ECs from WT and EC-p53KO mice for 48 h after irradiation. Scale bar = 50 μm (crop, 20 μm). Flow-cytometric analysis of CD206 + , iNOS + , and EdU + cells among F4/80 + cells (magnification, ×200) is shown. For ( d – f ), error bars indicate SEM; * p

    Article Snippet: RNA-seq analysis Total RNA was isolated from HUVECs, and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies).

    Techniques: RNA Sequencing Assay, Irradiation, Generated, Migration, Immunofluorescence, Mouse Assay, Flow Cytometry, Cytometry, Staining, Derivative Assay

    Increased OPN expression during radiation-induced EndMT correlates with proliferating CD44v6 + cancer cells in hypoxic areas. a – c HUVECs were transfected with siRNAs targeting TGFβR2 , Trp53 , or TGF β R2 + Trp53 for 48 h and irradiated (10 Gy). After 48 h in culture, total RNA was isolated for RNA-seq and RT-qPCR analyses. a Heat map of RNA-seq analysis showing inhibition of radiation-induced EndMT and the mesenchymal phenotype in HUVECs by Trp53 knockdown. b RT-qPCR analysis of EC-adhesion molecules, collagen, and fibroblastic markers in HUVECs. c RT-qPCR analysis of OPN in HUVECs. d Human soluble-receptor array analysis of conditioned medium from human pulmonary microvascular endothelial cells at 8 days after 5 Gy irradiation. The cyan boxes mark the spots corresponding to OPN. e , f Immunofluorescence detection of CD31 and OPN ( e ) or CD44v6, OPN, and CD31 ( f ) in KP tumours from irradiated WT and EC-p53KO mice (23 dpi). Scale bar = 20 μm. g Quantification of OPN + αSMA + CD31 + cells in the total CD31 + area (left) and OPN + CD44v6 + cells in the total CD44v6 + area (right) (magnification, ×200; n > 5). Mouse strain designations are the same as in Figs 1 and 2 . IR irradiation (20 Gy). h , i KP tumour cells were cultured under normoxia, 1% O 2 (mild hypoxia), or 0.5% O 2 (severe hypoxia) and irradiated with or without OPN. Data are representative of three independent experiments. h Immunofluorescence detection of CD44v6 and Ki67 in KP tumour cells 11 days after irradiation (left) and quantification of CD44v6 + Ki67 + cells in total CD44v6 + cells (right) (magnification, ×200; n > 5). Scale bar = 50 μm. i Flow-cytometric analysis of EdU incorporation and CD44v6 expression in KP tumour cells 11 days after irradiation. For ( b – d ), the error bars indicate SD of three independent experiments. For ( g – i ), the error bars indicate SEM; For ( b, c ), si-TGFβR2 + IR or si-Control + IR versus si-Control group, # p

    Journal: Nature Communications

    Article Title: Tumour-vasculature development via endothelial-to-mesenchymal transition after radiotherapy controls CD44v6+ cancer cell and macrophage polarization

    doi: 10.1038/s41467-018-07470-w

    Figure Lengend Snippet: Increased OPN expression during radiation-induced EndMT correlates with proliferating CD44v6 + cancer cells in hypoxic areas. a – c HUVECs were transfected with siRNAs targeting TGFβR2 , Trp53 , or TGF β R2 + Trp53 for 48 h and irradiated (10 Gy). After 48 h in culture, total RNA was isolated for RNA-seq and RT-qPCR analyses. a Heat map of RNA-seq analysis showing inhibition of radiation-induced EndMT and the mesenchymal phenotype in HUVECs by Trp53 knockdown. b RT-qPCR analysis of EC-adhesion molecules, collagen, and fibroblastic markers in HUVECs. c RT-qPCR analysis of OPN in HUVECs. d Human soluble-receptor array analysis of conditioned medium from human pulmonary microvascular endothelial cells at 8 days after 5 Gy irradiation. The cyan boxes mark the spots corresponding to OPN. e , f Immunofluorescence detection of CD31 and OPN ( e ) or CD44v6, OPN, and CD31 ( f ) in KP tumours from irradiated WT and EC-p53KO mice (23 dpi). Scale bar = 20 μm. g Quantification of OPN + αSMA + CD31 + cells in the total CD31 + area (left) and OPN + CD44v6 + cells in the total CD44v6 + area (right) (magnification, ×200; n > 5). Mouse strain designations are the same as in Figs 1 and 2 . IR irradiation (20 Gy). h , i KP tumour cells were cultured under normoxia, 1% O 2 (mild hypoxia), or 0.5% O 2 (severe hypoxia) and irradiated with or without OPN. Data are representative of three independent experiments. h Immunofluorescence detection of CD44v6 and Ki67 in KP tumour cells 11 days after irradiation (left) and quantification of CD44v6 + Ki67 + cells in total CD44v6 + cells (right) (magnification, ×200; n > 5). Scale bar = 50 μm. i Flow-cytometric analysis of EdU incorporation and CD44v6 expression in KP tumour cells 11 days after irradiation. For ( b – d ), the error bars indicate SD of three independent experiments. For ( g – i ), the error bars indicate SEM; For ( b, c ), si-TGFβR2 + IR or si-Control + IR versus si-Control group, # p

    Article Snippet: RNA-seq analysis Total RNA was isolated from HUVECs, and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies).

    Techniques: Expressing, Transfection, Irradiation, Isolation, RNA Sequencing Assay, Quantitative RT-PCR, Inhibition, Immunofluorescence, Mouse Assay, Cell Culture, Flow Cytometry

    Comprehensive genomic analyses of single-cell clones. ( A ) Genomic proportion of copy number alteration. For clonal samples B–F, > 84% of their genome is between copy number 3 and 4 (ranging from 84.82% to 85.9%, shown as dark grey bars) as compared to ≤3% for the other samples. This global whole genome duplication typically affected both alleles, preserving heterozygosity in these samples. ( B ) Number of somatic structural variants identified per sample with the type indicated by colours. ( C ) Hierarchical clustering of somatic substitution variant allele frequencies across tumour and clonal samples. The absence of a variant in the normal control cortex sample is demonstrated by a variant allele frequency of zero (dark blue). ( D ) Hierarchical clustering of Log2-normalised and gene-scaled RNA seq gene expression of genes with most variable expression as identified as contributing to the first principle component. Positive values indicate a sample with the highest expression and negative values with the lowest expression. ( E ) Hierarchical clustering of 1000 most variable β-values from DNA methylation sequencing data. Β-value of 1 indicates completely methylated and 0 unmethylated CpGs.

    Journal: Cancers

    Article Title: Intratumoural Heterogeneity Underlies Distinct Therapy Responses and Treatment Resistance in Glioblastoma

    doi: 10.3390/cancers11020190

    Figure Lengend Snippet: Comprehensive genomic analyses of single-cell clones. ( A ) Genomic proportion of copy number alteration. For clonal samples B–F, > 84% of their genome is between copy number 3 and 4 (ranging from 84.82% to 85.9%, shown as dark grey bars) as compared to ≤3% for the other samples. This global whole genome duplication typically affected both alleles, preserving heterozygosity in these samples. ( B ) Number of somatic structural variants identified per sample with the type indicated by colours. ( C ) Hierarchical clustering of somatic substitution variant allele frequencies across tumour and clonal samples. The absence of a variant in the normal control cortex sample is demonstrated by a variant allele frequency of zero (dark blue). ( D ) Hierarchical clustering of Log2-normalised and gene-scaled RNA seq gene expression of genes with most variable expression as identified as contributing to the first principle component. Positive values indicate a sample with the highest expression and negative values with the lowest expression. ( E ) Hierarchical clustering of 1000 most variable β-values from DNA methylation sequencing data. Β-value of 1 indicates completely methylated and 0 unmethylated CpGs.

    Article Snippet: DNA and RNA samples were quantified using Qubit Fluorometric Quantification instrument, and RNA integrity was assessed using RNA 6000 Nano Eukaryote Total RNA kit (Agilent Technologies cat. 5067-1511, Santa Clara, CA, USA) and Bioanalyzer 2100 instrument (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Clone Assay, Preserving, Variant Assay, RNA Sequencing Assay, Expressing, DNA Methylation Assay, Sequencing, Methylation

    Identification of miR-29b-1-5p-regulated genes. a Expression profiles of messenger RNAs (mRNAs) in K562 cells transfected with miR-29b-1-5p mimic or scramble oligonucleotides then cultured with medium for 48 h were evaluated using RNA-Seq transcriptome analysis. Each scatter spot represents the mean raw signals of microRNA (miRNA) in three repeats of each treatment. b The RNA expression levels of 16 genes were significantly higher, whereas the RNA expression levels of 10 genes were significantly lower, in K562 cells after being transfected with miR-29b-1-5p mimic for 48 h. c Four genes potentially involved in the inflammatory responses were selected for further analysis. The mRNA expression levels of ANG were significantly elevated in K562 cells after coculture with interleukin (IL)-23 (20 ng/ml) for 3 days (Fig. 3 c)

    Journal: Arthritis Research & Therapy

    Article Title: Aberrant expression of interleukin-23-regulated miRNAs in T cells from patients with ankylosing spondylitis

    doi: 10.1186/s13075-018-1754-1

    Figure Lengend Snippet: Identification of miR-29b-1-5p-regulated genes. a Expression profiles of messenger RNAs (mRNAs) in K562 cells transfected with miR-29b-1-5p mimic or scramble oligonucleotides then cultured with medium for 48 h were evaluated using RNA-Seq transcriptome analysis. Each scatter spot represents the mean raw signals of microRNA (miRNA) in three repeats of each treatment. b The RNA expression levels of 16 genes were significantly higher, whereas the RNA expression levels of 10 genes were significantly lower, in K562 cells after being transfected with miR-29b-1-5p mimic for 48 h. c Four genes potentially involved in the inflammatory responses were selected for further analysis. The mRNA expression levels of ANG were significantly elevated in K562 cells after coculture with interleukin (IL)-23 (20 ng/ml) for 3 days (Fig. 3 c)

    Article Snippet: Microarray analysis of miRNAs Total RNA (0.1 μg) was dephosphorylated and labeled with pCp-Cy3 by using the Agilent miRNA Complete Labeling and Hyb Kit (Agilent Technologies).

    Techniques: Expressing, Transfection, Cell Culture, RNA Sequencing Assay, RNA Expression