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    Millipore ribonucleic acid rna
    Log–log scatter plot depicting the normalized average difference (Avg Diff) intensity values for all present called probe sets used to monitor expression in E.coli when grown to mid log phase in rich LB medium. Diagonal lines in the graph represent 2-, 3-, 5- and 10-old variation between the compared expression experiments. ( A ) Scatter plot comparing the levels of expression in duplicate experiments using the <t>cDNA</t> sample preparation method and ( B ) scatter plot comparing the levels of expression in duplicate experiments using the direct labeling of enriched <t>RNA</t> method. ( C ) Scatter plot comparing the expression levels from the direct labeled enriched RNA method with the cDNA sample.
    Ribonucleic Acid Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Log–log scatter plot depicting the normalized average difference (Avg Diff) intensity values for all present called probe sets used to monitor expression in E.coli when grown to mid log phase in rich LB medium. Diagonal lines in the graph represent 2-, 3-, 5- and 10-old variation between the compared expression experiments. ( A ) Scatter plot comparing the levels of expression in duplicate experiments using the cDNA sample preparation method and ( B ) scatter plot comparing the levels of expression in duplicate experiments using the direct labeling of enriched RNA method. ( C ) Scatter plot comparing the expression levels from the direct labeled enriched RNA method with the cDNA sample.

    Journal: Nucleic Acids Research

    Article Title: Prokaryotic RNA preparation methods useful for high density array analysis: comparison of two approaches

    doi:

    Figure Lengend Snippet: Log–log scatter plot depicting the normalized average difference (Avg Diff) intensity values for all present called probe sets used to monitor expression in E.coli when grown to mid log phase in rich LB medium. Diagonal lines in the graph represent 2-, 3-, 5- and 10-old variation between the compared expression experiments. ( A ) Scatter plot comparing the levels of expression in duplicate experiments using the cDNA sample preparation method and ( B ) scatter plot comparing the levels of expression in duplicate experiments using the direct labeling of enriched RNA method. ( C ) Scatter plot comparing the expression levels from the direct labeled enriched RNA method with the cDNA sample.

    Article Snippet: Equal amounts of total RNA and cDNA (1 µg) were treated with a mixture of 1× SSC, 50% formamide and 6.48% formaldehyde (Sigma, St Louis, MO) in a volume of 40 µl.

    Techniques: Expressing, Sample Prep, Labeling

    Slot blot analysis of selected genes. Total RNA and synthesized cDNA was spotted in equivalent amounts (1 µg). After hybridization with a labeled PCR fragment and staining, the intensities were measured. The relative change in intensities is shown as the fold change.

    Journal: Nucleic Acids Research

    Article Title: Prokaryotic RNA preparation methods useful for high density array analysis: comparison of two approaches

    doi:

    Figure Lengend Snippet: Slot blot analysis of selected genes. Total RNA and synthesized cDNA was spotted in equivalent amounts (1 µg). After hybridization with a labeled PCR fragment and staining, the intensities were measured. The relative change in intensities is shown as the fold change.

    Article Snippet: Equal amounts of total RNA and cDNA (1 µg) were treated with a mixture of 1× SSC, 50% formamide and 6.48% formaldehyde (Sigma, St Louis, MO) in a volume of 40 µl.

    Techniques: Dot Blot, Synthesized, Hybridization, Labeling, Polymerase Chain Reaction, Staining

    Effect of ZFP57 loss on gene expression and genomic imprinting in inbred and hybrid ESCs, determined by global and allele-specific RNA-seq analyses. ( A ) Empirical cumulative distribution of the distances (bp) of deregulated genes (all genes, black; imprinted genes, green; non imprinted genes, orange) from ZFP57 binding sites in both Zfp57 −/− E14 and JB1 ESCs. ( B ) Scatter plot showing deregulated genes in both Zfp57 −/− E14 and JB1 ESCs. Deregulated genes are indicated by red dots if distant

    Journal: Nucleic Acids Research

    Article Title: ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells

    doi: 10.1093/nar/gkw505

    Figure Lengend Snippet: Effect of ZFP57 loss on gene expression and genomic imprinting in inbred and hybrid ESCs, determined by global and allele-specific RNA-seq analyses. ( A ) Empirical cumulative distribution of the distances (bp) of deregulated genes (all genes, black; imprinted genes, green; non imprinted genes, orange) from ZFP57 binding sites in both Zfp57 −/− E14 and JB1 ESCs. ( B ) Scatter plot showing deregulated genes in both Zfp57 −/− E14 and JB1 ESCs. Deregulated genes are indicated by red dots if distant

    Article Snippet: RNA analysis RNA was extracted from cultured ESCs by lysing the cells on the culture dish with the addition of the TRI reagent (Sigma-Aldrich) and following the protocol of the manufacturer.

    Techniques: Expressing, RNA Sequencing Assay, Binding Assay

    LOC100288798 exon structure assembly from various tissues extends its annotation to over 500kb overlapping SLC38A4 .UCSC Genome Browser screen shot of the studied locus (chr12:46,772,500-47,422,500). From top to bottom: Chromosome position and the scale; RefSeq gene annotation (all annotated isoforms are displayed), spliced human ESTs (12/35 ESTs displayed), transcriptome assembly of the locus obtained in this study (Results, Methods). Note that only selected transcripts are shown (11/167 de novo isoforms of LOC100288798 and 4/43 de novo isoforms of SLC38A4 ), and that both EST and transcriptome assembly data reveal extension of LOC100288798 to over 500kb in length. RNA-seq tracks from ENCODE/CSHL UCSC hub with the titles containing cell type name, RNA-seq type and transcriptional orientation are displayed below. Only total whole cell RNA-seq is displayed. Bottom: normalized RNA-seq signal from wild type human haploid KBM7 cell lines (merged data from 2 wild type clones sequenced in this study, Methods). For all RNA-seq tracks: only forward strand (Plus Signal) is displayed.

    Journal: RNA Biology

    Article Title: A human haploid gene trap collection to study lncRNAs with unusual RNA biology

    doi: 10.1080/15476286.2015.1110676

    Figure Lengend Snippet: LOC100288798 exon structure assembly from various tissues extends its annotation to over 500kb overlapping SLC38A4 .UCSC Genome Browser screen shot of the studied locus (chr12:46,772,500-47,422,500). From top to bottom: Chromosome position and the scale; RefSeq gene annotation (all annotated isoforms are displayed), spliced human ESTs (12/35 ESTs displayed), transcriptome assembly of the locus obtained in this study (Results, Methods). Note that only selected transcripts are shown (11/167 de novo isoforms of LOC100288798 and 4/43 de novo isoforms of SLC38A4 ), and that both EST and transcriptome assembly data reveal extension of LOC100288798 to over 500kb in length. RNA-seq tracks from ENCODE/CSHL UCSC hub with the titles containing cell type name, RNA-seq type and transcriptional orientation are displayed below. Only total whole cell RNA-seq is displayed. Bottom: normalized RNA-seq signal from wild type human haploid KBM7 cell lines (merged data from 2 wild type clones sequenced in this study, Methods). For all RNA-seq tracks: only forward strand (Plus Signal) is displayed.

    Article Snippet: RNA preparation RNA was isolated from pelleted KBM7 cells using TRIreagent (Sigma), dissolved in RNA Storage Solution (RSS, Ambion) and stored at −20o C. RNA was DNAse I treated (DNAfree kit, Ambion).

    Techniques: RNA Sequencing Assay, Clone Assay

    Haploid gene trap collection represents a rich resource for quick functional assessment of hundreds of lncRNAs. (A) Hundreds of GENCODE v19 lncRNAs expressed in KBM7 cell line are targeted by a gene trap insertion. Bar plot shows number of non-overlapping GENCODE v19 lncRNA loci that contain a gene trap cassette in the same transcriptional orientation in KBM7 clones within the “Human Gene Trap Mutant Collection” (left bar, Methods), and the number of these lncRNA loci that are expressed (middle bar, loci that contain lncRNA transcripts expressed with RPKM > 0.2) and well expressed (right bar, loci that contain lncRNA transcripts expressed with RPKM > 0.5) in wild type KBM7 cells. (B) Gene trap cassettes are preferentially inserted at the 5’ end of lncRNAs. Bar plot shows the number of gene trap cassettes inserted into different regions in the gene bodies of GENCODE v19 lncRNA. Numbers correspond to 10 equally sized, non-overlapping regions investigated for each gene. (C) Five genetic truncations of the well-known lncRNA MALAT1 are available within the “Human Gene Trap Mutant Collection.

    Journal: RNA Biology

    Article Title: A human haploid gene trap collection to study lncRNAs with unusual RNA biology

    doi: 10.1080/15476286.2015.1110676

    Figure Lengend Snippet: Haploid gene trap collection represents a rich resource for quick functional assessment of hundreds of lncRNAs. (A) Hundreds of GENCODE v19 lncRNAs expressed in KBM7 cell line are targeted by a gene trap insertion. Bar plot shows number of non-overlapping GENCODE v19 lncRNA loci that contain a gene trap cassette in the same transcriptional orientation in KBM7 clones within the “Human Gene Trap Mutant Collection” (left bar, Methods), and the number of these lncRNA loci that are expressed (middle bar, loci that contain lncRNA transcripts expressed with RPKM > 0.2) and well expressed (right bar, loci that contain lncRNA transcripts expressed with RPKM > 0.5) in wild type KBM7 cells. (B) Gene trap cassettes are preferentially inserted at the 5’ end of lncRNAs. Bar plot shows the number of gene trap cassettes inserted into different regions in the gene bodies of GENCODE v19 lncRNA. Numbers correspond to 10 equally sized, non-overlapping regions investigated for each gene. (C) Five genetic truncations of the well-known lncRNA MALAT1 are available within the “Human Gene Trap Mutant Collection." Shown is the UCSC browser screen shot of the MALAT1 gene region. From top to bottom: chromosome scale, CpG island annotation (UCSC track), FANTOM5 TSS predictions (robust set) 82 on the plus strand, RefSeq gene annotation, position of gene trap insertion cassettes available (plus strand), normalized RNA-seq signal from WT2 KBM7 cell line showing wild type expression of MALAT1 .

    Article Snippet: RNA preparation RNA was isolated from pelleted KBM7 cells using TRIreagent (Sigma), dissolved in RNA Storage Solution (RSS, Ambion) and stored at −20o C. RNA was DNAse I treated (DNAfree kit, Ambion).

    Techniques: Functional Assay, Clone Assay, Mutagenesis, RNA Sequencing Assay, Expressing

    Time courses for the formation of SAH (blue triangles), 5’-dA (red circles), ms 2 i 6 A (black triangles) and d 3 -ms 2 i 6 A (green triangles) upon ( A ) initial incubation of 150 μM Tm MiaB with a stoichiometric concentration of SAM in the absence of dithionite followed by ( B ) introduction of excess (500 μM) [ methyl - d 3 ]-SAM, 130 μM i 6 A ACSL RNA, and 1 mM dithionite in 50 mM Tris-HCl pH 7.5. The lines are fits to a first-order single-exponential equation, with the following obtained kinetic parameters: ( A ) SAH formation: A = 110 ± 4 μM, ν = 8.5 ± 1.3 min −1 ( B ) 5’-dA formation: A = 105 ± 3 μM, ν = 5.7 ± 0.7 μM min −1 ; SAH formation: A = 230 ± 3 μM, ν = 9.7 ± 0.8 μM min −1 ; ms 2 i 6 A formation: A = 40 ± 1 μM, ν = 11.6 ± 2.6 μM min −1 ; d 3 -ms 2 i 6 A formation: A = 48 ± 2 μM, ν = 1.3 ± 0.2 μM min −1 .

    Journal: Journal of the American Chemical Society

    Article Title: Identification of an Intermediate Methyl Carrier in the Radical SAM Methylthiotransferases, RimO and MiaB

    doi: 10.1021/ja4048448

    Figure Lengend Snippet: Time courses for the formation of SAH (blue triangles), 5’-dA (red circles), ms 2 i 6 A (black triangles) and d 3 -ms 2 i 6 A (green triangles) upon ( A ) initial incubation of 150 μM Tm MiaB with a stoichiometric concentration of SAM in the absence of dithionite followed by ( B ) introduction of excess (500 μM) [ methyl - d 3 ]-SAM, 130 μM i 6 A ACSL RNA, and 1 mM dithionite in 50 mM Tris-HCl pH 7.5. The lines are fits to a first-order single-exponential equation, with the following obtained kinetic parameters: ( A ) SAH formation: A = 110 ± 4 μM, ν = 8.5 ± 1.3 min −1 ( B ) 5’-dA formation: A = 105 ± 3 μM, ν = 5.7 ± 0.7 μM min −1 ; SAH formation: A = 230 ± 3 μM, ν = 9.7 ± 0.8 μM min −1 ; ms 2 i 6 A formation: A = 40 ± 1 μM, ν = 11.6 ± 2.6 μM min −1 ; d 3 -ms 2 i 6 A formation: A = 48 ± 2 μM, ν = 1.3 ± 0.2 μM min −1 .

    Article Snippet: The ACSL RNA was determined to be completely modified by MiaA, with each mol of RNA containing one mol of i6 A. Quantification was conducted by LC/MS using a standard curve generated from commercially available i6 A (Sigma).

    Techniques: Mass Spectrometry, Incubation, Concentration Assay

    Isotopic distribution of ms 2 i 6 A in assays containing 20 μM Tm MiaB, 100 μM i 6 A ACSL RNA substrate, and 1 mM dithionite in the presence of: ( A ) 500 μM SAM; ( B ) 500 μM d 3 -SAM and 500 μM NaSCH 3 ; and ( C ) 500 μM d 3 -SAM. After 2 h at 37 °C, ( A ) 19.4 μM ms 2 i 6 A was generated in the presence of SAM only ( B ) 11.7 μM ms 2 i 6 A and 9.8 μM d 3 -ms 2 i 6 A were generated in the presence both of SAM and NaSCH 3 , and ( C ) 21.2 μM d 3 -ms 2 i 6 A was generated in the presence of d 3 -SAM only.

    Journal: Journal of the American Chemical Society

    Article Title: Identification of an Intermediate Methyl Carrier in the Radical SAM Methylthiotransferases, RimO and MiaB

    doi: 10.1021/ja4048448

    Figure Lengend Snippet: Isotopic distribution of ms 2 i 6 A in assays containing 20 μM Tm MiaB, 100 μM i 6 A ACSL RNA substrate, and 1 mM dithionite in the presence of: ( A ) 500 μM SAM; ( B ) 500 μM d 3 -SAM and 500 μM NaSCH 3 ; and ( C ) 500 μM d 3 -SAM. After 2 h at 37 °C, ( A ) 19.4 μM ms 2 i 6 A was generated in the presence of SAM only ( B ) 11.7 μM ms 2 i 6 A and 9.8 μM d 3 -ms 2 i 6 A were generated in the presence both of SAM and NaSCH 3 , and ( C ) 21.2 μM d 3 -ms 2 i 6 A was generated in the presence of d 3 -SAM only.

    Article Snippet: The ACSL RNA was determined to be completely modified by MiaA, with each mol of RNA containing one mol of i6 A. Quantification was conducted by LC/MS using a standard curve generated from commercially available i6 A (Sigma).

    Techniques: Mass Spectrometry, Generated

    Preparation of Cas9 RNA and sgRNAs

    Journal: Development (Cambridge, England)

    Article Title: Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system

    doi: 10.1242/dev.115584

    Figure Lengend Snippet: Preparation of Cas9 RNA and sgRNAs

    Article Snippet: Preparation of Cas9 RNA and sgRNAs PCR amplifications were performed with KOD Hot Start DNA polymerase (Novagen).

    Techniques:

    Effects of TGF-β1 on expression of collagen III and fibronectin mRNA in HPMCs . Serum-starved HPMCs were incubated with TGF-β1 (2 or 10 ng/ml) for up to 72 h and RNA was then isolated and subjected to semi-quantitative RT-PCR analysis of collagen III (A) and fibronectin (B). Expression of β-actin was used as a loading control.

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Induction of gastric cancer cell adhesion through transforming growth factor-beta1-mediated peritoneal fibrosis

    doi: 10.1186/1756-9966-29-139

    Figure Lengend Snippet: Effects of TGF-β1 on expression of collagen III and fibronectin mRNA in HPMCs . Serum-starved HPMCs were incubated with TGF-β1 (2 or 10 ng/ml) for up to 72 h and RNA was then isolated and subjected to semi-quantitative RT-PCR analysis of collagen III (A) and fibronectin (B). Expression of β-actin was used as a loading control.

    Article Snippet: One microgram of the total cellular RNA was then reverse-transcribed into cDNA for PCR amplification using a kit from Sigma.

    Techniques: Expressing, Incubation, Isolation, Quantitative RT-PCR

    Raman imaging of typical human ESCs and iPSCs. Color-reconstituted Raman images of human embryonic stem cells (hESCs, upper left panel) and human induced pluripotent stem cells (hiPSCs) (upper right panel). White scale bar = 5 μm. Small insets show corresponding bright-field images recorded after Raman scanning. Raman peak at 785 cm –1 (DNA/RNA bases) mapped in red, 1670 cm –1 (proteins) in blue, 2850 cm –1 (lipids) in green, and a combination of 748 and 1585 cm –1 (cytochrome C) in magenta. hiPSCs exhibit a much higher level of DNA/RNA bases in well-defined regions of the cell. Curves in the lower panel are average spectra of hESCs (top curve) and hiPSCs (bottom curve), where the peaks used for the color-reconstituted images are indicated with the corresponding color

    Journal: Stem Cell Research & Therapy

    Article Title: Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy

    doi: 10.1186/s13287-017-0720-1

    Figure Lengend Snippet: Raman imaging of typical human ESCs and iPSCs. Color-reconstituted Raman images of human embryonic stem cells (hESCs, upper left panel) and human induced pluripotent stem cells (hiPSCs) (upper right panel). White scale bar = 5 μm. Small insets show corresponding bright-field images recorded after Raman scanning. Raman peak at 785 cm –1 (DNA/RNA bases) mapped in red, 1670 cm –1 (proteins) in blue, 2850 cm –1 (lipids) in green, and a combination of 748 and 1585 cm –1 (cytochrome C) in magenta. hiPSCs exhibit a much higher level of DNA/RNA bases in well-defined regions of the cell. Curves in the lower panel are average spectra of hESCs (top curve) and hiPSCs (bottom curve), where the peaks used for the color-reconstituted images are indicated with the corresponding color

    Article Snippet: DNA and RNA analyses for nucleic acid quantification and gel electrophoresis Genomic DNA (gDNA) from hiPSCs and hESCs was extracted using a GenElute Mammalian Genomic DNA Miniprep kit (Sigma Aldrich, Saint Louis, MO, USA), while total RNA was extracted using an Absolutely RNA Miniprep kit (Agilent Technologies).

    Techniques: Imaging

    Principal component (PC) curves as biochemical indicators. a Loading curves of the first three PCs calculated by PCA. Since PCA is performed on the overall dataset of all probed cells, the computed PCs are the same throughout all of the measured spectra, and their characteristic bands indicate sensitive biochemical features (see text for details concerning peak assignment). While the PC1 curve only resembles the global average spectrum, the PC2 and PC3 curves account for the significant biochemical differences between the different cells, as well as different regions of the same cell. Scatter plot of PC3 vs PC2 scores for hESCs (blue dots) and hiPSCs (red dots) from Fig. 1 . Each dot corresponds to one spectrum (pixel) of Raman mapping. Blue and red dots closely overlap, except for the top-left part of the graph which corresponds to positive PC3 and negative PC2 scores ( b ). Loading curves of ( a ) support that this PC3–PC2 region can be assigned to DNA/RNA bases, the typical frequencies of which are exhibited as positive bands in the PC3 curve (mainly 785, 1098, 1484, and 1575 cm –1 ), and as a sharp negative band (785 cm –1 ) for the PC2 curve. hESC human embryonic stem cell, hiPSC human induced pluripotent stem cell

    Journal: Stem Cell Research & Therapy

    Article Title: Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy

    doi: 10.1186/s13287-017-0720-1

    Figure Lengend Snippet: Principal component (PC) curves as biochemical indicators. a Loading curves of the first three PCs calculated by PCA. Since PCA is performed on the overall dataset of all probed cells, the computed PCs are the same throughout all of the measured spectra, and their characteristic bands indicate sensitive biochemical features (see text for details concerning peak assignment). While the PC1 curve only resembles the global average spectrum, the PC2 and PC3 curves account for the significant biochemical differences between the different cells, as well as different regions of the same cell. Scatter plot of PC3 vs PC2 scores for hESCs (blue dots) and hiPSCs (red dots) from Fig. 1 . Each dot corresponds to one spectrum (pixel) of Raman mapping. Blue and red dots closely overlap, except for the top-left part of the graph which corresponds to positive PC3 and negative PC2 scores ( b ). Loading curves of ( a ) support that this PC3–PC2 region can be assigned to DNA/RNA bases, the typical frequencies of which are exhibited as positive bands in the PC3 curve (mainly 785, 1098, 1484, and 1575 cm –1 ), and as a sharp negative band (785 cm –1 ) for the PC2 curve. hESC human embryonic stem cell, hiPSC human induced pluripotent stem cell

    Article Snippet: DNA and RNA analyses for nucleic acid quantification and gel electrophoresis Genomic DNA (gDNA) from hiPSCs and hESCs was extracted using a GenElute Mammalian Genomic DNA Miniprep kit (Sigma Aldrich, Saint Louis, MO, USA), while total RNA was extracted using an Absolutely RNA Miniprep kit (Agilent Technologies).

    Techniques:

    Quantification of nucleic acid levels. DNA and RNA extracted from both pluripotent stem cell lines were quantified with a NanoDrop 2000 UV-Vis spectrophotometer ( a ) and with ethidium bromide staining on agarose gel electrophoresis ( b ). Error bars indicate mean ± SEM. Statistical comparison between hiPSCs and hESCs by paired Student’s t test (* p

    Journal: Stem Cell Research & Therapy

    Article Title: Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy

    doi: 10.1186/s13287-017-0720-1

    Figure Lengend Snippet: Quantification of nucleic acid levels. DNA and RNA extracted from both pluripotent stem cell lines were quantified with a NanoDrop 2000 UV-Vis spectrophotometer ( a ) and with ethidium bromide staining on agarose gel electrophoresis ( b ). Error bars indicate mean ± SEM. Statistical comparison between hiPSCs and hESCs by paired Student’s t test (* p

    Article Snippet: DNA and RNA analyses for nucleic acid quantification and gel electrophoresis Genomic DNA (gDNA) from hiPSCs and hESCs was extracted using a GenElute Mammalian Genomic DNA Miniprep kit (Sigma Aldrich, Saint Louis, MO, USA), while total RNA was extracted using an Absolutely RNA Miniprep kit (Agilent Technologies).

    Techniques: Spectrophotometry, Staining, Agarose Gel Electrophoresis

    Semiquantitative comparison of Raman images by cluster analysis. KCA performed on the PCA results for Raman assignment of different cellular regions. Top row reports results for three typical hESCs, while second row reports results for three typical hiPSCs (scale bar = 5 μm). For the KCA calculation, six clusters were imposed within the cells (see text for further details), and the red cluster is only evident in hiPSCs (upper panel). Lower graph shows average Raman spectra of each cluster, where the curves have the same color as the corresponding cluster. The red curve exhibits all of the major peaks ascribed to DNA/RNA bases, and consequently the red regions inside the cells are assigned to DNA/RNA compartments. The absence of red clusters inside hESCs does not mean that DNA/RNA bases are missing therein, only that their expression is much lower than the DNA/RNA abundance in the red regions of hiPSCs

    Journal: Stem Cell Research & Therapy

    Article Title: Two sides of the same coin? Unraveling subtle differences between human embryonic and induced pluripotent stem cells by Raman spectroscopy

    doi: 10.1186/s13287-017-0720-1

    Figure Lengend Snippet: Semiquantitative comparison of Raman images by cluster analysis. KCA performed on the PCA results for Raman assignment of different cellular regions. Top row reports results for three typical hESCs, while second row reports results for three typical hiPSCs (scale bar = 5 μm). For the KCA calculation, six clusters were imposed within the cells (see text for further details), and the red cluster is only evident in hiPSCs (upper panel). Lower graph shows average Raman spectra of each cluster, where the curves have the same color as the corresponding cluster. The red curve exhibits all of the major peaks ascribed to DNA/RNA bases, and consequently the red regions inside the cells are assigned to DNA/RNA compartments. The absence of red clusters inside hESCs does not mean that DNA/RNA bases are missing therein, only that their expression is much lower than the DNA/RNA abundance in the red regions of hiPSCs

    Article Snippet: DNA and RNA analyses for nucleic acid quantification and gel electrophoresis Genomic DNA (gDNA) from hiPSCs and hESCs was extracted using a GenElute Mammalian Genomic DNA Miniprep kit (Sigma Aldrich, Saint Louis, MO, USA), while total RNA was extracted using an Absolutely RNA Miniprep kit (Agilent Technologies).

    Techniques: Expressing

    Transcriptional analysis of RNA recovered from wild type and allelic exchange mutant E . chaffeensis organisms assessed by RT-PCR. ( A ) RT-PCR products from wild type (W) and Ech_0230 mutant (M) organisms were resolved (L, 1 kb plus molecular weight DNA markers resolved; +, genomic DNA from wild type E . chaffeensis was used as the template; -, negative control reaction with no template added). ( B ) As in panel A, except that the analysis was performed using RNA recovered from Ech_0379 disruption (M) and restoration (R) mutant organisms. Positive controls for this experiments included genomic DNAs as the templates from W, M and R. (0.38 kb amplicons are expected for DNA templates in PCRs of W and R and 1.6 kb product is expected for M DNA as the template.) ( C ) Mutations to inactivate and restore the gene activity in Ech_0379 did not alter the gene expression from its neighboring genes. Semi-quantitative RT-PCR assays were performed at 30, 35 and 40 PCR cycles for Ech_0378, Ech_0379 and Ech_0380 for wild type, gene inactivation and gene rescue mutant organisms and the data for 35 cycles were presented. W, M and R had similar quantities of amplicons for Ech_0378 and Ech_0380; Ech_0379 amplicons were also similar for W and R, while absent for M. (Full-length gels and blots were included in the Supplementary Figure file, as parts of the Figure had cropped images).

    Journal: Scientific Reports

    Article Title: A genetic system for targeted mutations to disrupt and restore genes in the obligate bacterium, Ehrlichia chaffeensis

    doi: 10.1038/s41598-017-16023-y

    Figure Lengend Snippet: Transcriptional analysis of RNA recovered from wild type and allelic exchange mutant E . chaffeensis organisms assessed by RT-PCR. ( A ) RT-PCR products from wild type (W) and Ech_0230 mutant (M) organisms were resolved (L, 1 kb plus molecular weight DNA markers resolved; +, genomic DNA from wild type E . chaffeensis was used as the template; -, negative control reaction with no template added). ( B ) As in panel A, except that the analysis was performed using RNA recovered from Ech_0379 disruption (M) and restoration (R) mutant organisms. Positive controls for this experiments included genomic DNAs as the templates from W, M and R. (0.38 kb amplicons are expected for DNA templates in PCRs of W and R and 1.6 kb product is expected for M DNA as the template.) ( C ) Mutations to inactivate and restore the gene activity in Ech_0379 did not alter the gene expression from its neighboring genes. Semi-quantitative RT-PCR assays were performed at 30, 35 and 40 PCR cycles for Ech_0378, Ech_0379 and Ech_0380 for wild type, gene inactivation and gene rescue mutant organisms and the data for 35 cycles were presented. W, M and R had similar quantities of amplicons for Ech_0378 and Ech_0380; Ech_0379 amplicons were also similar for W and R, while absent for M. (Full-length gels and blots were included in the Supplementary Figure file, as parts of the Figure had cropped images).

    Article Snippet: RNA analysis by RT-PCR to verify the loss and restoration of transcription Total RNAs from wild type and mutant E . chaffeensis organisms grown in ISE6 or DH82 cell cultures were isolated by following the Tri-reagent total RNA isolation method (Sigma-Aldrich, St. Louis, MO).

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Negative Control, Activity Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction