Structured Review

Agilent technologies bioanalyzer
VELCRO-IP RT-qPCR serves as a proof-of-principle to identify novel hES9S-interacting 5’ UTRs and mRNA fragmentation. Related to Figure 3. (A) Schematic of in vitro transcripts used for the proof-of-principle experiment of the VELCRO-IP RT-qPCR. Reproduced from Figure 3B . (B) For qualitative analysis of the integrity of in vitro transcripts, RNAs were subjected to 4-20% polyacrylamide/TBE/native PAGE and visualized by SYBR Gold staining. (C) Analysis of total RNA in the 3xFlag peptide elution by RT-qPCR using same volumes of RNA per sample for the RT. Normalization of Ct values for Fluc to the 18S rRNA tag internally controls for ribosome-IP efficiency per sample. The native/WT sample was used to normalize for fold enrichment of RNA binding (set to 1). Representation of the raw data in Figure 3D . Average RNA fold enrichment, SEM, n = 5; ns, not significant. (D) Full view of the <t>Bioanalyzer</t> (Agilent) quantification and electronic gel analysis in Figure 3G, H is shown for optimization of mouse mRNA fragmentation from C3H/10T1/2 cells and stage E11.5 mouse embryos. The marker (M, grey) is overlaid for reference. (E) Full view of the Bioanalyzer (Agilent) quantification and electronic gel analysis in Figure 4B is shown for the eluted and yeast rRNA-depleted mouse embryo RNA from three independent replicates of WT and hES9S VELCRO-IP experiments. The marker (M, grey) is overlaid for reference.
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1) Product Images from "VELCRO-IP RNA-seq explores ribosome expansion segment function in translation genome-wide"

Article Title: VELCRO-IP RNA-seq explores ribosome expansion segment function in translation genome-wide

Journal: bioRxiv

doi: 10.1101/2020.07.01.179515

VELCRO-IP RT-qPCR serves as a proof-of-principle to identify novel hES9S-interacting 5’ UTRs and mRNA fragmentation. Related to Figure 3. (A) Schematic of in vitro transcripts used for the proof-of-principle experiment of the VELCRO-IP RT-qPCR. Reproduced from Figure 3B . (B) For qualitative analysis of the integrity of in vitro transcripts, RNAs were subjected to 4-20% polyacrylamide/TBE/native PAGE and visualized by SYBR Gold staining. (C) Analysis of total RNA in the 3xFlag peptide elution by RT-qPCR using same volumes of RNA per sample for the RT. Normalization of Ct values for Fluc to the 18S rRNA tag internally controls for ribosome-IP efficiency per sample. The native/WT sample was used to normalize for fold enrichment of RNA binding (set to 1). Representation of the raw data in Figure 3D . Average RNA fold enrichment, SEM, n = 5; ns, not significant. (D) Full view of the Bioanalyzer (Agilent) quantification and electronic gel analysis in Figure 3G, H is shown for optimization of mouse mRNA fragmentation from C3H/10T1/2 cells and stage E11.5 mouse embryos. The marker (M, grey) is overlaid for reference. (E) Full view of the Bioanalyzer (Agilent) quantification and electronic gel analysis in Figure 4B is shown for the eluted and yeast rRNA-depleted mouse embryo RNA from three independent replicates of WT and hES9S VELCRO-IP experiments. The marker (M, grey) is overlaid for reference.
Figure Legend Snippet: VELCRO-IP RT-qPCR serves as a proof-of-principle to identify novel hES9S-interacting 5’ UTRs and mRNA fragmentation. Related to Figure 3. (A) Schematic of in vitro transcripts used for the proof-of-principle experiment of the VELCRO-IP RT-qPCR. Reproduced from Figure 3B . (B) For qualitative analysis of the integrity of in vitro transcripts, RNAs were subjected to 4-20% polyacrylamide/TBE/native PAGE and visualized by SYBR Gold staining. (C) Analysis of total RNA in the 3xFlag peptide elution by RT-qPCR using same volumes of RNA per sample for the RT. Normalization of Ct values for Fluc to the 18S rRNA tag internally controls for ribosome-IP efficiency per sample. The native/WT sample was used to normalize for fold enrichment of RNA binding (set to 1). Representation of the raw data in Figure 3D . Average RNA fold enrichment, SEM, n = 5; ns, not significant. (D) Full view of the Bioanalyzer (Agilent) quantification and electronic gel analysis in Figure 3G, H is shown for optimization of mouse mRNA fragmentation from C3H/10T1/2 cells and stage E11.5 mouse embryos. The marker (M, grey) is overlaid for reference. (E) Full view of the Bioanalyzer (Agilent) quantification and electronic gel analysis in Figure 4B is shown for the eluted and yeast rRNA-depleted mouse embryo RNA from three independent replicates of WT and hES9S VELCRO-IP experiments. The marker (M, grey) is overlaid for reference.

Techniques Used: Quantitative RT-PCR, In Vitro, Clear Native PAGE, Staining, RNA Binding Assay, Marker

VELCRO-IP RNA-seq identifies global ES-mRNA interactions with positional resolution on mRNAs. (A) For VELCRO-IP RNA-seq, mRNA was isolated from stage E11.5 mouse embryos, fragmented to 100-200 nt and used as input for the IP. Total RNA obtained from all samples after elution and yeast rRNA depletion obtains ribosome-bound mouse mRNA that was subjected to library preparation and Illumina high-throughput sequencing (NextSeq). We included an mRNA fragment input sample for reference. The distribution of mRNA fragment lengths for all sequenced libraries is plotted and the median fragment length is 246 nt. We then map all reads to the mouse and yeast transcriptomes and only further analyze reads exclusively mapping to mouse mRNAs. (B) Eluted and yeast rRNA-depleted mouse RNA from three independent replicates of WT and hES9S VELCRO-IP experiments were analyzed on a mRNA Pico Chip (Agilent) on a Bioanalyzer (Agilent) and a zoomed-in view of the Bioanalyzer quantification and electronic gel analysis is shown. The marker (M, grey) is overlaid for reference. See also Figure S3E . (C) WB analysis as in Figure 3C to monitor efficient IP of 40S ribosomes for RNA-seq of mouse mRNA fragments. Representative of n = 3 is shown. (D) Kernel density of the distribution of t-statistics for test of differential enrichment of mRNA fragments bound to hES9S vs WT ribosomes is plotted in black. Empirical estimate of the decomposition of the distribution to null and non-null tests are plotted in grey and red, respectively. Dotted line indicates where the local FDR of 0.05 is. (E) RNA-seq results of independent replicates (n = 3) for each WT and hES9S samples. Normalized log read counts are presented for WT and hES9S-enriched mRNA fragments. Fragments less than FDR
Figure Legend Snippet: VELCRO-IP RNA-seq identifies global ES-mRNA interactions with positional resolution on mRNAs. (A) For VELCRO-IP RNA-seq, mRNA was isolated from stage E11.5 mouse embryos, fragmented to 100-200 nt and used as input for the IP. Total RNA obtained from all samples after elution and yeast rRNA depletion obtains ribosome-bound mouse mRNA that was subjected to library preparation and Illumina high-throughput sequencing (NextSeq). We included an mRNA fragment input sample for reference. The distribution of mRNA fragment lengths for all sequenced libraries is plotted and the median fragment length is 246 nt. We then map all reads to the mouse and yeast transcriptomes and only further analyze reads exclusively mapping to mouse mRNAs. (B) Eluted and yeast rRNA-depleted mouse RNA from three independent replicates of WT and hES9S VELCRO-IP experiments were analyzed on a mRNA Pico Chip (Agilent) on a Bioanalyzer (Agilent) and a zoomed-in view of the Bioanalyzer quantification and electronic gel analysis is shown. The marker (M, grey) is overlaid for reference. See also Figure S3E . (C) WB analysis as in Figure 3C to monitor efficient IP of 40S ribosomes for RNA-seq of mouse mRNA fragments. Representative of n = 3 is shown. (D) Kernel density of the distribution of t-statistics for test of differential enrichment of mRNA fragments bound to hES9S vs WT ribosomes is plotted in black. Empirical estimate of the decomposition of the distribution to null and non-null tests are plotted in grey and red, respectively. Dotted line indicates where the local FDR of 0.05 is. (E) RNA-seq results of independent replicates (n = 3) for each WT and hES9S samples. Normalized log read counts are presented for WT and hES9S-enriched mRNA fragments. Fragments less than FDR

Techniques Used: RNA Sequencing Assay, Isolation, Next-Generation Sequencing, Chromatin Immunoprecipitation, Marker, Western Blot

2) Product Images from "COBRA-Seq: Sensitive and Quantitative Methylome Profiling"

Article Title: COBRA-Seq: Sensitive and Quantitative Methylome Profiling

Journal: Genes

doi: 10.3390/genes6041140

GW-COBRA and LA-COBRA library construction results. ( A ) Sonicated genomic DNA isolated from HCT116 cell line (lane 1), 50 ng of Adapter-2 ligated library material (lane 2). Column purified Adapter-2 ligated library material (lane 3); ( B ) PCR amplification test of bisulfite treated library materials. Lane 1, 2, 3 and 4 are produced by 6, 7, 8 and 9 cycles of PCR using GW-A2-FwdP and COBRA-A2-RevP primers from Table 1 with GW-COBRA library material as the template respectively. Lane 5, 6, 7 and 8 are produced by 6, 7, 8 and 9 cycles of PCR using LA-A2+T7-FwdP and COBRA-A2-RevP primers from Table 1 with LA-COBRA library material as the template, respectively; ( C ) Final library products: GW-COBRA and LA-COBRA methylome libraries of HCT116 DNA amplified using flowcell or LADS primer pairs respectively (Lane 2 and 4). Lane 1 and 3 are negative controls for GW-COBRA and LA-COBRA libraries, respectively; ( D ) Bioanalyzer results of GW-COBRA and LA-COBRA methylome sequencing libraries, respectively, prepared from HCT116 cell line DNA. The final library fragments ranged between 150–500 bp with an average size of 257 and 360 bp for GW-COBRA and LA-COBRA, respectively.
Figure Legend Snippet: GW-COBRA and LA-COBRA library construction results. ( A ) Sonicated genomic DNA isolated from HCT116 cell line (lane 1), 50 ng of Adapter-2 ligated library material (lane 2). Column purified Adapter-2 ligated library material (lane 3); ( B ) PCR amplification test of bisulfite treated library materials. Lane 1, 2, 3 and 4 are produced by 6, 7, 8 and 9 cycles of PCR using GW-A2-FwdP and COBRA-A2-RevP primers from Table 1 with GW-COBRA library material as the template respectively. Lane 5, 6, 7 and 8 are produced by 6, 7, 8 and 9 cycles of PCR using LA-A2+T7-FwdP and COBRA-A2-RevP primers from Table 1 with LA-COBRA library material as the template, respectively; ( C ) Final library products: GW-COBRA and LA-COBRA methylome libraries of HCT116 DNA amplified using flowcell or LADS primer pairs respectively (Lane 2 and 4). Lane 1 and 3 are negative controls for GW-COBRA and LA-COBRA libraries, respectively; ( D ) Bioanalyzer results of GW-COBRA and LA-COBRA methylome sequencing libraries, respectively, prepared from HCT116 cell line DNA. The final library fragments ranged between 150–500 bp with an average size of 257 and 360 bp for GW-COBRA and LA-COBRA, respectively.

Techniques Used: Combined Bisulfite Restriction Analysis Assay, Sonication, Isolation, Purification, Polymerase Chain Reaction, Amplification, Produced, Sequencing

3) Product Images from "Identification of N6,N6-Dimethyladenosine in Transfer RNA from Mycobacterium bovis Bacille Calmette-Guérin"

Article Title: Identification of N6,N6-Dimethyladenosine in Transfer RNA from Mycobacterium bovis Bacille Calmette-Guérin

Journal: Molecules

doi: 10.3390/molecules16065168

Characterization of BCG small RNA species. An aliquot of small RNA isolated from BCG was analyzed on an Agilent Bioanalyzer small RNA chip. The peak at 4 nt in the electropherogram represents a size standard; the image on the right is the reconstructed gel image of the resolved RNA species.
Figure Legend Snippet: Characterization of BCG small RNA species. An aliquot of small RNA isolated from BCG was analyzed on an Agilent Bioanalyzer small RNA chip. The peak at 4 nt in the electropherogram represents a size standard; the image on the right is the reconstructed gel image of the resolved RNA species.

Techniques Used: Isolation, Chromatin Immunoprecipitation

4) Product Images from "Early dynamics of photosynthetic Lhcf2 and Lhcf15 transcription and mRNA stabilities in response to herbivory-related decadienal in Phaeodactylum tricornutum"

Article Title: Early dynamics of photosynthetic Lhcf2 and Lhcf15 transcription and mRNA stabilities in response to herbivory-related decadienal in Phaeodactylum tricornutum

Journal: Scientific Reports

doi: 10.1038/s41598-020-58885-9

In vivo incorporation of 4-TU into RNA of P. tricornutum . ( a ) Schematic workflow of 4-TU labeling into RNA and detection. Bioanalyzer profiles of RNA fractions from cell cultures treated with 0.5 mM 4-TU ( b ) or 0.1% DMSO as solvent control ( c ) for 1.5 h. From each treated culture, total RNA represents the biotinylated RNA fraction and eluted RNA represents the newly synthesized RNA fraction. Equal masses of biotinylated RNA fractions were used to prepare the eluted RNA fractions.
Figure Legend Snippet: In vivo incorporation of 4-TU into RNA of P. tricornutum . ( a ) Schematic workflow of 4-TU labeling into RNA and detection. Bioanalyzer profiles of RNA fractions from cell cultures treated with 0.5 mM 4-TU ( b ) or 0.1% DMSO as solvent control ( c ) for 1.5 h. From each treated culture, total RNA represents the biotinylated RNA fraction and eluted RNA represents the newly synthesized RNA fraction. Equal masses of biotinylated RNA fractions were used to prepare the eluted RNA fractions.

Techniques Used: In Vivo, Labeling, Synthesized

5) Product Images from "Translational reprogramming of colorectal cancer cells induced by 5-fluorouracil through a miRNA-dependent mechanism"

Article Title: Translational reprogramming of colorectal cancer cells induced by 5-fluorouracil through a miRNA-dependent mechanism

Journal: Oncotarget

doi: 10.18632/oncotarget.17597

Impact of 5-FU treatment on protein synthesis in HCT-116 cells (A-B) Global protein synthesis in response to 5-FU. Protein synthesis was quantified by 35 S labeling pulse-chase assays in non-treated and 5-FU treated cells. A representative gel is shown in (A) and mean quantification of three independent experiments is shown in (B). Compared to non-treated cells, a reproducible decrease in protein synthesis was observed in response to 10 μM of 5-FU for 24 hrs. Cycloheximide (CHX) was used as a positive control. (C-D) Polysome profiles in response to 5-FU. 40S and 60S ribosomal subunits, 80S monosomes and polysomes were separated by ultracentrifugation on sucrose gradients. One representative polysome profile of non-treated (C) and 10 μM 5-FU treated cells (D) is shown, as well as gel analysis of 18S and 28S rRNA used to verified RNA quality. On top of each profile, the fractions collected for microarray analyses (non-polysome NP and polysome P) are indicated. After RNA extraction, RNA quality was checked using bioanalyzer, the RNA Integrity Number (RIN) ranging from 6.6 to 9.3.
Figure Legend Snippet: Impact of 5-FU treatment on protein synthesis in HCT-116 cells (A-B) Global protein synthesis in response to 5-FU. Protein synthesis was quantified by 35 S labeling pulse-chase assays in non-treated and 5-FU treated cells. A representative gel is shown in (A) and mean quantification of three independent experiments is shown in (B). Compared to non-treated cells, a reproducible decrease in protein synthesis was observed in response to 10 μM of 5-FU for 24 hrs. Cycloheximide (CHX) was used as a positive control. (C-D) Polysome profiles in response to 5-FU. 40S and 60S ribosomal subunits, 80S monosomes and polysomes were separated by ultracentrifugation on sucrose gradients. One representative polysome profile of non-treated (C) and 10 μM 5-FU treated cells (D) is shown, as well as gel analysis of 18S and 28S rRNA used to verified RNA quality. On top of each profile, the fractions collected for microarray analyses (non-polysome NP and polysome P) are indicated. After RNA extraction, RNA quality was checked using bioanalyzer, the RNA Integrity Number (RIN) ranging from 6.6 to 9.3.

Techniques Used: Labeling, Pulse Chase, Positive Control, Microarray, RNA Extraction

6) Product Images from "Horizontal transfer of exosomal microRNAs transduce apoptotic signals between pancreatic beta-cells"

Article Title: Horizontal transfer of exosomal microRNAs transduce apoptotic signals between pancreatic beta-cells

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/s12964-015-0097-7

Characterization of exosomes released by MIN6B1 cells. A) The sizes of microvesicles isolated by ultra-centrifugation from the culture media of MIN6B1 cells was measured using the Nanosight technology. B, C) Three different preparations of microvesicles from MIN6B1 cells were analyzed by immunoblotting for B) the presence of the exosomal protein markers Alix, CD81 and Tsg101, and C) for the absence of the cellular protein Calnexin. Cell extract was used as positive control for the detection of Calnexin. D) RNA profiles of MIN6B1 cells (upper panel) and of microvesicles (lower panel) were determined by Bioanalyzer.
Figure Legend Snippet: Characterization of exosomes released by MIN6B1 cells. A) The sizes of microvesicles isolated by ultra-centrifugation from the culture media of MIN6B1 cells was measured using the Nanosight technology. B, C) Three different preparations of microvesicles from MIN6B1 cells were analyzed by immunoblotting for B) the presence of the exosomal protein markers Alix, CD81 and Tsg101, and C) for the absence of the cellular protein Calnexin. Cell extract was used as positive control for the detection of Calnexin. D) RNA profiles of MIN6B1 cells (upper panel) and of microvesicles (lower panel) were determined by Bioanalyzer.

Techniques Used: Isolation, Centrifugation, Positive Control

7) Product Images from "Exploring genome wide bisulfite sequencing for DNA methylation analysis in livestock: a technical assessment"

Article Title: Exploring genome wide bisulfite sequencing for DNA methylation analysis in livestock: a technical assessment

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2014.00126

Bioanalyzer gel image of the three RRBS libraries made with different insert sizes . Ligated adapters cause the DNA fragments to migrate to a higher molecular weight (approximately 100 bp higher) than the insert sizes selected.
Figure Legend Snippet: Bioanalyzer gel image of the three RRBS libraries made with different insert sizes . Ligated adapters cause the DNA fragments to migrate to a higher molecular weight (approximately 100 bp higher) than the insert sizes selected.

Techniques Used: Molecular Weight

8) Product Images from "The mysterious Spotted Green Pigeon and its relation to the Dodo and its kindred"

Article Title: The mysterious Spotted Green Pigeon and its relation to the Dodo and its kindred

Journal: BMC Evolutionary Biology

doi: 10.1186/1471-2148-14-136

The Spotted Green Pigeon, extracted DNA characteristics and phylogeny. (A) Reconstruction of the Spotted Green or Liverpool Pigeon (courtesy of del Hoyo, J., Elliott, A., Sargatal, J. eds. 2002. Handbook of the Birds of the World. Vol. 7. Jacamars to Woodpeckers. Lynx Edicions, Barcelona), (B) a picture of the sole surviving specimen (courtesy of Clemency Fisher and the World Museum, National Museums Liverpool), (C) Bioanalyzer plot for the first DNA extract highlighting the short fragmentary nature of the DNA (median 51 bp). 35 bp and 10380 bp peaks are markers. FU: fluorescent units., (D) Maximum likelihood tree for the concatenated Spotted Green Pigeon sequences and 12S sequences from members of the extended Dodo clade (as identified by Shapiro et al. [ 1 ]) and (E) Maximum likelihood tree for 106 Pigeon mitochondrial 12S sequences. The Spotted Green Pigeon (bold) clusters first with the Nicobar Pigeon and second with the Dodo and Rodrigues Solitaire. Previously identified Pigeon clades in the phylogeny are coloured. The reason for the clustering of three rock Pigeon sequences ( Columba livia , grey box) with mourning doves ( Zenaida macroura ) is unclear, although hybridisation has been observed for these two species [ 5 ]. Bootstrap support values above 40 are indicated in the larger tree to allow for observation of the bootstrap value for the split between Caloenas and Raphinae, the dotted lines in both trees are there to associate the taxa with the appropriate tree tips.
Figure Legend Snippet: The Spotted Green Pigeon, extracted DNA characteristics and phylogeny. (A) Reconstruction of the Spotted Green or Liverpool Pigeon (courtesy of del Hoyo, J., Elliott, A., Sargatal, J. eds. 2002. Handbook of the Birds of the World. Vol. 7. Jacamars to Woodpeckers. Lynx Edicions, Barcelona), (B) a picture of the sole surviving specimen (courtesy of Clemency Fisher and the World Museum, National Museums Liverpool), (C) Bioanalyzer plot for the first DNA extract highlighting the short fragmentary nature of the DNA (median 51 bp). 35 bp and 10380 bp peaks are markers. FU: fluorescent units., (D) Maximum likelihood tree for the concatenated Spotted Green Pigeon sequences and 12S sequences from members of the extended Dodo clade (as identified by Shapiro et al. [ 1 ]) and (E) Maximum likelihood tree for 106 Pigeon mitochondrial 12S sequences. The Spotted Green Pigeon (bold) clusters first with the Nicobar Pigeon and second with the Dodo and Rodrigues Solitaire. Previously identified Pigeon clades in the phylogeny are coloured. The reason for the clustering of three rock Pigeon sequences ( Columba livia , grey box) with mourning doves ( Zenaida macroura ) is unclear, although hybridisation has been observed for these two species [ 5 ]. Bootstrap support values above 40 are indicated in the larger tree to allow for observation of the bootstrap value for the split between Caloenas and Raphinae, the dotted lines in both trees are there to associate the taxa with the appropriate tree tips.

Techniques Used: Hybridization

9) Product Images from "RNA sequencing data of Notch ligand treated human dental pulp cells"

Article Title: RNA sequencing data of Notch ligand treated human dental pulp cells

Journal: Data in Brief

doi: 10.1016/j.dib.2018.01.058

Quality check of input total RNA using the Bioanalyzer. (A-C) hFc replicates; (D-F) Jagged1 replicates; (G-I) Jagged1+DAPT replicates.
Figure Legend Snippet: Quality check of input total RNA using the Bioanalyzer. (A-C) hFc replicates; (D-F) Jagged1 replicates; (G-I) Jagged1+DAPT replicates.

Techniques Used:

10) Product Images from "Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding"

Article Title: Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

Journal: Scientific Reports

doi: 10.1038/srep44447

Bulk validation of SOE-PCR linkage of antibody and cell barcode sequences. ( a ) Generation of SOE-PCR product depends on the presence of both antibody tag and cell barcode sequences, as demonstrated on a 1% agarose gel stained with SYBR green. ( b ) The SOE-PCR product is pure, yielding a sharp peak on a Bioanalyzer at the anticipated molecular weight. ( c ) For Abseq to provide quantitative results, the number of SOE-PCR products must be in proportion to the number of antibody tag sequences bound to the cell, which we validate by quantitative PCR. When the appropriate antibody is used, amplification occurs early, indicating presence of much SOE-PCR product (green and red). When no or the incorrect antibody is used, amplification occurs late, indicating little SOE-PCR product.
Figure Legend Snippet: Bulk validation of SOE-PCR linkage of antibody and cell barcode sequences. ( a ) Generation of SOE-PCR product depends on the presence of both antibody tag and cell barcode sequences, as demonstrated on a 1% agarose gel stained with SYBR green. ( b ) The SOE-PCR product is pure, yielding a sharp peak on a Bioanalyzer at the anticipated molecular weight. ( c ) For Abseq to provide quantitative results, the number of SOE-PCR products must be in proportion to the number of antibody tag sequences bound to the cell, which we validate by quantitative PCR. When the appropriate antibody is used, amplification occurs early, indicating presence of much SOE-PCR product (green and red). When no or the incorrect antibody is used, amplification occurs late, indicating little SOE-PCR product.

Techniques Used: Overlap Extension Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, SYBR Green Assay, Molecular Weight, Real-time Polymerase Chain Reaction, Amplification

11) Product Images from "Cross-Contamination of a UROtsa Stock with T24 Cells - Molecular Comparison of Different Cell Lines and Stocks"

Article Title: Cross-Contamination of a UROtsa Stock with T24 Cells - Molecular Comparison of Different Cell Lines and Stocks

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064139

PCR analysis of cell lines and UROtsa stocks to detect large T-antigen. (A) PCR reactions were resolved on a 2% agarose gel. SV40gp6 sequences (5′-, 3′-, and middle part) are absent in UROtsa-1 (lanes 2, 10, 18), UROtsa-3/T24 (lanes 3, 11, 19), UROtsa-4 (lanes 4, 12, 20), HeLa (negative control, lanes 5, 13, 21) and T24 (negative control, lanes 6, 14, 22) but not in BEAS-2B (positive control, lanes 7, 15, 23). No-template controls are in lanes 1, 9, and 17. A 100 bp ladder served as size marker (lanes 8, 16, 24). (B) PCR reactions of reverse-transcribed mRNA were resolved on a microfluidic DNA 1000 chip (Bioanalyzer). Large T-antigen expression (expected product size: 65 bp in lanes 2–7 [24] and 304 bp in lanes 8–13 [23] ) is absent in UROtsa-3/T24 (lanes 3 and 8), UROtsa-4 (lanes 4 and 9), HeLa (negative control, lanes 5 and 10), and T24 (negative control, lanes 6 and 11) but not in BEAS-2B (positive control, lanes 7 and 12). A no-template control was loaded in lanes 2 and 13. Size markers (lane 1) are 15 (green), 25, 50, 100, 150, 200, 300, 400, 500, 700, 850, 1000, and 1500 bp (purple).
Figure Legend Snippet: PCR analysis of cell lines and UROtsa stocks to detect large T-antigen. (A) PCR reactions were resolved on a 2% agarose gel. SV40gp6 sequences (5′-, 3′-, and middle part) are absent in UROtsa-1 (lanes 2, 10, 18), UROtsa-3/T24 (lanes 3, 11, 19), UROtsa-4 (lanes 4, 12, 20), HeLa (negative control, lanes 5, 13, 21) and T24 (negative control, lanes 6, 14, 22) but not in BEAS-2B (positive control, lanes 7, 15, 23). No-template controls are in lanes 1, 9, and 17. A 100 bp ladder served as size marker (lanes 8, 16, 24). (B) PCR reactions of reverse-transcribed mRNA were resolved on a microfluidic DNA 1000 chip (Bioanalyzer). Large T-antigen expression (expected product size: 65 bp in lanes 2–7 [24] and 304 bp in lanes 8–13 [23] ) is absent in UROtsa-3/T24 (lanes 3 and 8), UROtsa-4 (lanes 4 and 9), HeLa (negative control, lanes 5 and 10), and T24 (negative control, lanes 6 and 11) but not in BEAS-2B (positive control, lanes 7 and 12). A no-template control was loaded in lanes 2 and 13. Size markers (lane 1) are 15 (green), 25, 50, 100, 150, 200, 300, 400, 500, 700, 850, 1000, and 1500 bp (purple).

Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control, Positive Control, Marker, Chromatin Immunoprecipitation, Expressing

12) Product Images from "Reference gene selection for gene expression study in shell gland and spleen of laying hens challenged with infectious bronchitis virus"

Article Title: Reference gene selection for gene expression study in shell gland and spleen of laying hens challenged with infectious bronchitis virus

Journal: Scientific Reports

doi: 10.1038/s41598-017-14693-2

Amplification of the genes fragments from the shell gland tissue of chicken to assess the specificities of the primers used in the current study. (L) DNA ladder (bp); ( 1 ) 18 S rRNA (63 bp); ( 2 ) ALB (197 bp); ( 3 ) ACTB (139 bp); ( 4 ) GAPDH (66 bp); ( 5 ) HMBS (131 bp); ( 6 ) HPRT1 (245 bp); ( 7 ) RPL4 (235 bp); ( 8 ) SDHA (126 bp); ( 9 ) TBP (147 bp); ( 10 ) YWHAZ (61 bp); ( 11 ) ND4-positive control (137 bp); ( 12 ) TLR7-positive control (200 bp). The upper (purple) and lower (green) markers act as internal standards and are used to align the ladder analysis with the individual DNA sample analysis. The standard curve (plotting migration time against DNA amplicon size), in conjunction with the markers, is then used to calculate DNA fragment sizes for each well from the migration times measured (see Agilent 2100 Bioanalyzer Users Guide for Molecular Assays). The DNA gel in Agilent 2100 Bioanalyzer was performed as per manufacturer’s instructions of Agilent DNA 1000 Kit.
Figure Legend Snippet: Amplification of the genes fragments from the shell gland tissue of chicken to assess the specificities of the primers used in the current study. (L) DNA ladder (bp); ( 1 ) 18 S rRNA (63 bp); ( 2 ) ALB (197 bp); ( 3 ) ACTB (139 bp); ( 4 ) GAPDH (66 bp); ( 5 ) HMBS (131 bp); ( 6 ) HPRT1 (245 bp); ( 7 ) RPL4 (235 bp); ( 8 ) SDHA (126 bp); ( 9 ) TBP (147 bp); ( 10 ) YWHAZ (61 bp); ( 11 ) ND4-positive control (137 bp); ( 12 ) TLR7-positive control (200 bp). The upper (purple) and lower (green) markers act as internal standards and are used to align the ladder analysis with the individual DNA sample analysis. The standard curve (plotting migration time against DNA amplicon size), in conjunction with the markers, is then used to calculate DNA fragment sizes for each well from the migration times measured (see Agilent 2100 Bioanalyzer Users Guide for Molecular Assays). The DNA gel in Agilent 2100 Bioanalyzer was performed as per manufacturer’s instructions of Agilent DNA 1000 Kit.

Techniques Used: Amplification, Positive Control, Activated Clotting Time Assay, Migration

13) Product Images from "Genome-wide mapping of embedded ribonucleotides and other non-canonical nucleotides using emRiboSeq and EndoSeq"

Article Title: Genome-wide mapping of embedded ribonucleotides and other non-canonical nucleotides using emRiboSeq and EndoSeq

Journal: Nature protocols

doi: 10.1038/nprot.2015.099

Library quality control and anticipated results. ( a ) Sonicated DNA separated by agarose gel electrophoresis (Step 25) shows an average fragment size of approximately 400 bp. ( b ) Bioanalyzer result (Step 84) for an emRiboSeq library shows a typical trace (left) and gel-like image (right) with a peak for fragments between ˜180 and ˜300 bp in size (black bar). Standards (green and purple bars) of defined size and amount allow quantification. FU, arbitrary fluorescence units. ( c ) Agarose gel electrophoresis of PCR products after 15, 16 and 17 cycles of amplification (Steps 81-83) of the same library shows product between 200 and 300 bp in size. ( d ) Sequencing results for libraries generated using Nb.BtsI are highly reproducibility between different strains (POL, wildtype polymerase; pol1-L868M, increased Pol-α ribonucleotide incorporation) after normalizing read counts to sequence tags per million (TPM). The majority of bona fide Nb.BtsI sites were present at maximal frequency, although some sites were present at lower frequencies. This is the result of partial loss during size selection because of their close proximity to other cleavage sites, a highly reproducible finding between independent libraries (Spearman's rho=0.82, p
Figure Legend Snippet: Library quality control and anticipated results. ( a ) Sonicated DNA separated by agarose gel electrophoresis (Step 25) shows an average fragment size of approximately 400 bp. ( b ) Bioanalyzer result (Step 84) for an emRiboSeq library shows a typical trace (left) and gel-like image (right) with a peak for fragments between ˜180 and ˜300 bp in size (black bar). Standards (green and purple bars) of defined size and amount allow quantification. FU, arbitrary fluorescence units. ( c ) Agarose gel electrophoresis of PCR products after 15, 16 and 17 cycles of amplification (Steps 81-83) of the same library shows product between 200 and 300 bp in size. ( d ) Sequencing results for libraries generated using Nb.BtsI are highly reproducibility between different strains (POL, wildtype polymerase; pol1-L868M, increased Pol-α ribonucleotide incorporation) after normalizing read counts to sequence tags per million (TPM). The majority of bona fide Nb.BtsI sites were present at maximal frequency, although some sites were present at lower frequencies. This is the result of partial loss during size selection because of their close proximity to other cleavage sites, a highly reproducible finding between independent libraries (Spearman's rho=0.82, p

Techniques Used: Sonication, Agarose Gel Electrophoresis, Fluorescence, Polymerase Chain Reaction, Amplification, Sequencing, Generated, Selection

14) Product Images from "Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes"

Article Title: Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0128036

Correspondence between BioAnalyzer traces and the length distribution of aligned reads. Panels A-C show three representative BioAnalyzer traces from three sample preparations of S . maltophilia . Panels D-F show the corresponding estimated fragment-size distributions (black) and the actual distributions of fragment lengths imputed from alignment to the reference genome (blue). A BioAnalyzer trace f(x) shows fluorescence f at fragment length x . However, we are interested in n(x) , the (relative) number of fragments n of length x . Since fluorescence of a DNA fragment is proportional to its length, n(x) ∝ f(x) / x . Note that sequencing can be successful despite the presence of apparently very long fragments (which are likely heteroduplexes) in the BioAnalyzer traces (Panels C and F).
Figure Legend Snippet: Correspondence between BioAnalyzer traces and the length distribution of aligned reads. Panels A-C show three representative BioAnalyzer traces from three sample preparations of S . maltophilia . Panels D-F show the corresponding estimated fragment-size distributions (black) and the actual distributions of fragment lengths imputed from alignment to the reference genome (blue). A BioAnalyzer trace f(x) shows fluorescence f at fragment length x . However, we are interested in n(x) , the (relative) number of fragments n of length x . Since fluorescence of a DNA fragment is proportional to its length, n(x) ∝ f(x) / x . Note that sequencing can be successful despite the presence of apparently very long fragments (which are likely heteroduplexes) in the BioAnalyzer traces (Panels C and F).

Techniques Used: Fluorescence, Sequencing

Dependence of fragment size distribution on input gDNA concentration and bead volume. DNA fragment size distribution is affected by starting genomic DNA concentration (rows) as described in Module 1 as well as the relative amount of bead buffer used in PCR clean-up (columns) as described in Module 4. Size distribution is measured by BioAnalyzer and reported in fluorescence units. Data is from Stenotrophomonas maltophilia (67% GC, 4.8 Mb genome). At high initial gDNA concentration (1.25 ng/μl) the fragment distribution is right-skewed, though this anomalous peak does not appear to significantly affect sequencing output.
Figure Legend Snippet: Dependence of fragment size distribution on input gDNA concentration and bead volume. DNA fragment size distribution is affected by starting genomic DNA concentration (rows) as described in Module 1 as well as the relative amount of bead buffer used in PCR clean-up (columns) as described in Module 4. Size distribution is measured by BioAnalyzer and reported in fluorescence units. Data is from Stenotrophomonas maltophilia (67% GC, 4.8 Mb genome). At high initial gDNA concentration (1.25 ng/μl) the fragment distribution is right-skewed, though this anomalous peak does not appear to significantly affect sequencing output.

Techniques Used: Concentration Assay, Polymerase Chain Reaction, Fluorescence, Sequencing

15) Product Images from "Revelation of mRNAs and proteins in porcine milk exosomes by transcriptomic and proteomic analysis"

Article Title: Revelation of mRNAs and proteins in porcine milk exosomes by transcriptomic and proteomic analysis

Journal: BMC Veterinary Research

doi: 10.1186/s12917-017-1021-8

Identification of proteins and mRNAs in porcine milk exosomes. a detection of the exosomal marker proteins CD63 and CD9 by Western blotting. b SDS-PAGE. c RNA sample analyzed by the Agilent Bioanalyzer 2100. d distribution of genen’s coverage
Figure Legend Snippet: Identification of proteins and mRNAs in porcine milk exosomes. a detection of the exosomal marker proteins CD63 and CD9 by Western blotting. b SDS-PAGE. c RNA sample analyzed by the Agilent Bioanalyzer 2100. d distribution of genen’s coverage

Techniques Used: Marker, Western Blot, SDS Page

16) Product Images from "Adipose-Derived Stem Cells Induce Angiogenesis via Microvesicle Transport of miRNA-31"

Article Title: Adipose-Derived Stem Cells Induce Angiogenesis via Microvesicle Transport of miRNA-31

Journal: Stem Cells Translational Medicine

doi: 10.5966/sctm.2015-0177

Analysis of RNA extracted from ASC-released MVs. (A): Small RNA (fewer than 200 nucleotides) and large RNA extracted from ASCs and ASC-released MVs were quantified ( n = 3– 4). Total RNA was set to 100%. (B): Equal amounts of total RNA from ASCs and MVs were analyzed using Bioanalyzer (Agilent Technologies). 18S and 28S indicated the ribosomal RNA. (C): Small RNA from MVs was analyzed using Bioanalyzer. (D): An electropherogram of the lane MV on (C) . (E): Reverse-transcriptase-polymerase chain reaction analysis of miRNA for small RNA extracted from MV-P. The level of miRNA in MV was set to 1 (dashed line). U6 was used as an internal control. n = 4. ∗, p
Figure Legend Snippet: Analysis of RNA extracted from ASC-released MVs. (A): Small RNA (fewer than 200 nucleotides) and large RNA extracted from ASCs and ASC-released MVs were quantified ( n = 3– 4). Total RNA was set to 100%. (B): Equal amounts of total RNA from ASCs and MVs were analyzed using Bioanalyzer (Agilent Technologies). 18S and 28S indicated the ribosomal RNA. (C): Small RNA from MVs was analyzed using Bioanalyzer. (D): An electropherogram of the lane MV on (C) . (E): Reverse-transcriptase-polymerase chain reaction analysis of miRNA for small RNA extracted from MV-P. The level of miRNA in MV was set to 1 (dashed line). U6 was used as an internal control. n = 4. ∗, p

Techniques Used: Polymerase Chain Reaction

17) Product Images from "Inhibition of stationary phase respiration impairs persister formation in E. coli"

Article Title: Inhibition of stationary phase respiration impairs persister formation in E. coli

Journal: Nature communications

doi: 10.1038/ncomms8983

RNA integrity, protein levels and degradation, and cells size of stationary phase cells ( A–C, E–F ) Cell cultures at early stationary phase (t=6 h) were treated with 1mM KCN or transferred to an anaerobic chamber. At t=24 h, cells were pelleted for RNA, protein, and microscope analyses. For controls, untreated overnight cultures (t=24 h) and early stationary phase cultures (t=6 h) were used. ( A–B ) RNA quality was determined with a bioanalyzer using an RNA 6000 Nano kit. The degradation of rRNA was assessed with RNA integrity values which range from 10 (intact) to 1 (totally degraded). (C) Cells were sonicated and the protein content in the supernatant was determined with Bradford assays. (D) Before KCN treatment at t=6 h, the inducer for gfp expression was removed in the cultures with the cells carrying pQE-80L gfp-ssrA . After the KCN treatment, GFP levels were measured. Background fluorescence was determined using cells with empty vectors. (E–F) Phase contrast images of fixed cells were taken using a microscope, and cell size (fold change relative to 24 h untreated overnight cultures) were determined with ImageJ. (G–H) KCN treatment was performed at t=9 h. At t=24 h, microscope images were taken, and ampicillin and ofloxacin persister levels were determined. * signifies significant differences for comparisons to control groups (p-value
Figure Legend Snippet: RNA integrity, protein levels and degradation, and cells size of stationary phase cells ( A–C, E–F ) Cell cultures at early stationary phase (t=6 h) were treated with 1mM KCN or transferred to an anaerobic chamber. At t=24 h, cells were pelleted for RNA, protein, and microscope analyses. For controls, untreated overnight cultures (t=24 h) and early stationary phase cultures (t=6 h) were used. ( A–B ) RNA quality was determined with a bioanalyzer using an RNA 6000 Nano kit. The degradation of rRNA was assessed with RNA integrity values which range from 10 (intact) to 1 (totally degraded). (C) Cells were sonicated and the protein content in the supernatant was determined with Bradford assays. (D) Before KCN treatment at t=6 h, the inducer for gfp expression was removed in the cultures with the cells carrying pQE-80L gfp-ssrA . After the KCN treatment, GFP levels were measured. Background fluorescence was determined using cells with empty vectors. (E–F) Phase contrast images of fixed cells were taken using a microscope, and cell size (fold change relative to 24 h untreated overnight cultures) were determined with ImageJ. (G–H) KCN treatment was performed at t=9 h. At t=24 h, microscope images were taken, and ampicillin and ofloxacin persister levels were determined. * signifies significant differences for comparisons to control groups (p-value

Techniques Used: Microscopy, Sonication, Expressing, Fluorescence, Significance Assay

18) Product Images from "Downregulation of ALAS1 by nicarbazin treatment underlies the reduced synthesis of protoporphyrin IX in shell gland of laying hens"

Article Title: Downregulation of ALAS1 by nicarbazin treatment underlies the reduced synthesis of protoporphyrin IX in shell gland of laying hens

Journal: Scientific Reports

doi: 10.1038/s41598-017-06527-y

Amplification of the genes fragments from the shell gland tissue of a chicken to assess the specificities of the primers used in the current study. L. DNA marker; (1) ND4 (137 bp); (2) GAPDH (137 bp); (3) SLC25A38 (139 bp); (4) ALAS1 (197 bp); (5) ABCB6 (107 bp); (6) CPOX (187 bp); (7) ABCG2 (208 bp); (8) FECH (112 bp); (9). FLVCR1 (239 bp); (10) HMBS (131 bp); (11) HPRT1 (245 bp); (12) TLR7-positive control (200 bp). The upper (purple) and lower (green) markers act as internal standards and are used to align the ladder analysis with the individual DNA sample analysis. The standard curve (plotting migration time against DNA amplicon size), in conjunction with the markers, is then used to calculate DNA fragment sizes for each well from the migration times measured (see Agilent 2100 Bioanalyzer Users Guide for Molecular Assays). The DNA gel in Agilent 2100 Bioanalyzer was performed as per the manufacturer’s instructions of Agilent DNA 1000 Kit.
Figure Legend Snippet: Amplification of the genes fragments from the shell gland tissue of a chicken to assess the specificities of the primers used in the current study. L. DNA marker; (1) ND4 (137 bp); (2) GAPDH (137 bp); (3) SLC25A38 (139 bp); (4) ALAS1 (197 bp); (5) ABCB6 (107 bp); (6) CPOX (187 bp); (7) ABCG2 (208 bp); (8) FECH (112 bp); (9). FLVCR1 (239 bp); (10) HMBS (131 bp); (11) HPRT1 (245 bp); (12) TLR7-positive control (200 bp). The upper (purple) and lower (green) markers act as internal standards and are used to align the ladder analysis with the individual DNA sample analysis. The standard curve (plotting migration time against DNA amplicon size), in conjunction with the markers, is then used to calculate DNA fragment sizes for each well from the migration times measured (see Agilent 2100 Bioanalyzer Users Guide for Molecular Assays). The DNA gel in Agilent 2100 Bioanalyzer was performed as per the manufacturer’s instructions of Agilent DNA 1000 Kit.

Techniques Used: Amplification, Marker, Positive Control, Activated Clotting Time Assay, Migration

19) 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

20) Product Images from "A method for high‐throughput production of sequence‐verified DNA libraries and strain collections"

Article Title: A method for high‐throughput production of sequence‐verified DNA libraries and strain collections

Journal: Molecular Systems Biology

doi: 10.15252/msb.20167233

Assembly of the mCherry gene from sequence‐verified fragments (1) First, 14 yeast clones containing sequence‐perfect fragments for assembly of mCherry were combined at equal concentrations and genomic DNA was isolated from this mixture. (2) From this genomic DNA, the 14 fragments were PCR‐amplified using common priming sites. Below each section, a bioanalyzer trace shows that the fragments were all of the appropriate size. (3) After PCR, the common priming sites were removed by digestion with BTSa1. (4) The digested fragments were then PCR‐assembled with primers that added homologies for vector integration, and size‐selected to obtain fragments of the appropriate size. (5) The assembled mCherry fragment was then inserted into a linearized expression vector by yeast homologous recombination. Expression of mCherry was confirmed by microscopy. Phase contrast (left) and mCherry (right) images for BY4743 cells and BY4743 cells carrying the mCherry plasmid are shown. Sanger sequencing confirmed perfect DNA sequence in six out of six clones.
Figure Legend Snippet: Assembly of the mCherry gene from sequence‐verified fragments (1) First, 14 yeast clones containing sequence‐perfect fragments for assembly of mCherry were combined at equal concentrations and genomic DNA was isolated from this mixture. (2) From this genomic DNA, the 14 fragments were PCR‐amplified using common priming sites. Below each section, a bioanalyzer trace shows that the fragments were all of the appropriate size. (3) After PCR, the common priming sites were removed by digestion with BTSa1. (4) The digested fragments were then PCR‐assembled with primers that added homologies for vector integration, and size‐selected to obtain fragments of the appropriate size. (5) The assembled mCherry fragment was then inserted into a linearized expression vector by yeast homologous recombination. Expression of mCherry was confirmed by microscopy. Phase contrast (left) and mCherry (right) images for BY4743 cells and BY4743 cells carrying the mCherry plasmid are shown. Sanger sequencing confirmed perfect DNA sequence in six out of six clones.

Techniques Used: Sequencing, Clone Assay, Isolation, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Expressing, Homologous Recombination, Microscopy

21) Product Images from "Use of an Automated Multiple-Locus, Variable-Number Tandem Repeat-Based Method for Rapid and High-Throughput Genotyping of Staphylococcus aureus Isolates"

Article Title: Use of an Automated Multiple-Locus, Variable-Number Tandem Repeat-Based Method for Rapid and High-Throughput Genotyping of Staphylococcus aureus Isolates

Journal:

doi: 10.1128/JCM.43.7.3346-3355.2005

Comparison of electropherograms obtained for the strains sequenced. Representative electropherograms and the corresponding gel patterns (BioAnalyzer) obtained for strains MW2 and N315 are shown. Strain MW2 displays eight peaks, while strain N315 shows
Figure Legend Snippet: Comparison of electropherograms obtained for the strains sequenced. Representative electropherograms and the corresponding gel patterns (BioAnalyzer) obtained for strains MW2 and N315 are shown. Strain MW2 displays eight peaks, while strain N315 shows

Techniques Used:

Effect of lysis duration on DNA yields and PCR amplification results. (A) Amounts of total purified DNA obtained after the rapid lysis procedure (strain MW2), as determined by the fluorescence area under the curve (BioAnalyzer). The electropherogram shown
Figure Legend Snippet: Effect of lysis duration on DNA yields and PCR amplification results. (A) Amounts of total purified DNA obtained after the rapid lysis procedure (strain MW2), as determined by the fluorescence area under the curve (BioAnalyzer). The electropherogram shown

Techniques Used: Lysis, Polymerase Chain Reaction, Amplification, Purification, Fluorescence

22) Product Images from "Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types"

Article Title: Single-cell RNA sequencing of mouse brain and lung vascular and vessel-associated cell types

Journal: Scientific Data

doi: 10.1038/sdata.2018.160

Quality control of the single cell sequencing library preparation. ( a ) Representative cDNA graphs from a validation plate, analyzed with a High Sensitivity D5000 ScreenTape on a TapeStation 4200. The size distribution of the cDNA was established by running a ladder (top left). A1 and A2 represent positive controls (20 cells), while P1 and P2 are empty negative controls. The other graphs display cDNA size distribution of randomly picked single cells of a validation plate after amplification. Graph E1 has colored boxes to assist in clarifying peak significance: Red boxes indicate upper and lower markers for size selection, the green box shows the cDNA size distribution and the blue box highlights the primer dimers. ( b ) Quality control of the pooled sequencing library after tagmentation with homemade Tn5 and indexing with the Nextera Indexing XT kit. The sequencing pools were analyzed on a BioAnalyzer with a High Sensitivity DNA Chip. Colored boxes describe peak significance as described above.
Figure Legend Snippet: Quality control of the single cell sequencing library preparation. ( a ) Representative cDNA graphs from a validation plate, analyzed with a High Sensitivity D5000 ScreenTape on a TapeStation 4200. The size distribution of the cDNA was established by running a ladder (top left). A1 and A2 represent positive controls (20 cells), while P1 and P2 are empty negative controls. The other graphs display cDNA size distribution of randomly picked single cells of a validation plate after amplification. Graph E1 has colored boxes to assist in clarifying peak significance: Red boxes indicate upper and lower markers for size selection, the green box shows the cDNA size distribution and the blue box highlights the primer dimers. ( b ) Quality control of the pooled sequencing library after tagmentation with homemade Tn5 and indexing with the Nextera Indexing XT kit. The sequencing pools were analyzed on a BioAnalyzer with a High Sensitivity DNA Chip. Colored boxes describe peak significance as described above.

Techniques Used: Sequencing, Amplification, Selection, Chromatin Immunoprecipitation

23) Product Images from "Extraction of microRNAs from biological matrices with titanium dioxide nanofibers"

Article Title: Extraction of microRNAs from biological matrices with titanium dioxide nanofibers

Journal: Analytical and bioanalytical chemistry

doi: 10.1007/s00216-017-0649-3

Comparison of small RNA extraction from MDA-MB-231 cells with fibers and columns analyzed by an Agilent 2100 Bioanalyzer. Small RNA recovery was as high as 985 pg/μL with the fibers and 10.2 pg/μL with the columns from as little as 134,000
Figure Legend Snippet: Comparison of small RNA extraction from MDA-MB-231 cells with fibers and columns analyzed by an Agilent 2100 Bioanalyzer. Small RNA recovery was as high as 985 pg/μL with the fibers and 10.2 pg/μL with the columns from as little as 134,000

Techniques Used: RNA Extraction, Multiple Displacement Amplification

24) Product Images from "Comparison of DNA Quantification Methods for Next Generation Sequencing"

Article Title: Comparison of DNA Quantification Methods for Next Generation Sequencing

Journal: Scientific Reports

doi: 10.1038/srep24067

Comparison of four titration techniques for NGS. ( a ) Diagram of the Next generation sequencing (NGS) experimental design. Using four quantification methods, we titrated DNA libraries prepared from HeLa cells, following manufacturers’ instructions. Six different indexes were added at the amplification step. Of the eight lanes on the NGS flowcell, four lanes were used to compare each method (unique index lane) and four lanes for pooling accuracy (pool of six indexes per lane). ( b ) Bioanalyzer image of the libraries, showing a homogenous smear of DNA from 280 to 450 bp. All six indexes show similarly average sizes. ( c ) Result of the quantification for all indexes using QuBit, qPCR, ddPCR and ddPCR-Tail approaches, respectively. All quantifications (except ddPCR-Tail) were done following manufacturer instructions (Invitrogen, BioRad, and KapaBiosystem). The ddPCR-Tail strategy used the same apparatus as the ddPCR with slight modifications (50 nM primers; three-step PCR-annealing at 65 °C for 30 seconds, extension time of 30 seconds at 72 °C). Experiments done in triplicate, mean calculated using dilution curve (average of 12 values per quantification, 6 for QuBit). Mean ± SD shown.
Figure Legend Snippet: Comparison of four titration techniques for NGS. ( a ) Diagram of the Next generation sequencing (NGS) experimental design. Using four quantification methods, we titrated DNA libraries prepared from HeLa cells, following manufacturers’ instructions. Six different indexes were added at the amplification step. Of the eight lanes on the NGS flowcell, four lanes were used to compare each method (unique index lane) and four lanes for pooling accuracy (pool of six indexes per lane). ( b ) Bioanalyzer image of the libraries, showing a homogenous smear of DNA from 280 to 450 bp. All six indexes show similarly average sizes. ( c ) Result of the quantification for all indexes using QuBit, qPCR, ddPCR and ddPCR-Tail approaches, respectively. All quantifications (except ddPCR-Tail) were done following manufacturer instructions (Invitrogen, BioRad, and KapaBiosystem). The ddPCR-Tail strategy used the same apparatus as the ddPCR with slight modifications (50 nM primers; three-step PCR-annealing at 65 °C for 30 seconds, extension time of 30 seconds at 72 °C). Experiments done in triplicate, mean calculated using dilution curve (average of 12 values per quantification, 6 for QuBit). Mean ± SD shown.

Techniques Used: Titration, Next-Generation Sequencing, Amplification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Titration of six NGS libraries with low abundance with qPCR and ddPCR Tail. ( a ) Comparison of two methods for low abundance NGS libraries, titration results using qPCR and the ddPCR-Tail system. Assay done in triplicate and mean calculated using dilution correction factors (average of 18 values per sample). Mean ± SD shown. ( b ) Bioanalyzer image results used to calculate final molarity for the qPCR measurement, algorithm provided by KapaBiosystem using the average size of the NGS library combined with the qPCR standard curve. ( c ) All libraries were successfully quantified with high confidence (linear regression all in 0.9), ddPCR Tail strategy showed a better overall linearity regardless of the heterogeneity of the libraries.
Figure Legend Snippet: Titration of six NGS libraries with low abundance with qPCR and ddPCR Tail. ( a ) Comparison of two methods for low abundance NGS libraries, titration results using qPCR and the ddPCR-Tail system. Assay done in triplicate and mean calculated using dilution correction factors (average of 18 values per sample). Mean ± SD shown. ( b ) Bioanalyzer image results used to calculate final molarity for the qPCR measurement, algorithm provided by KapaBiosystem using the average size of the NGS library combined with the qPCR standard curve. ( c ) All libraries were successfully quantified with high confidence (linear regression all in 0.9), ddPCR Tail strategy showed a better overall linearity regardless of the heterogeneity of the libraries.

Techniques Used: Titration, Next-Generation Sequencing, Real-time Polymerase Chain Reaction

25) Product Images from "Determining degradation intermediates and the pathway of 3’ to 5’ degradation of histone mRNA using high-throughput sequencing"

Article Title: Determining degradation intermediates and the pathway of 3’ to 5’ degradation of histone mRNA using high-throughput sequencing

Journal: Methods (San Diego, Calif.)

doi: 10.1016/j.ymeth.2018.11.001

Quality Control of EnD-Seq Libraries. The final libraries are analyzed on a Agilent Bioanalyzer. About 10 ng of each sample (determined by the Cubit reading) is loaded in each lane. Shown is a standard DNA Agilent Chip. All the libraries we plan to multiplex are run on the same Chip. The expected products are about 450 nts for full-length histone mRNAs, with shorter lengths for the degradation intermediates. Primer dimers from PCR2 are about 120 nts and are a common contaminant. The left lane is the provided ladder for determining library size. Lanes 2, 4, 5, and 8 excellent libraries with minimal primer dimers. Lanes 1 and 6 have some primer dimer contamination, but gave good results on sequencing. Lanes 3 and 9 have excessive amounts of primer dimer. This is generally corrected by reamplifying the cDNA. Lane 7 is a failed library, which may require starting from a new reverse transcription of the ligated RNA.
Figure Legend Snippet: Quality Control of EnD-Seq Libraries. The final libraries are analyzed on a Agilent Bioanalyzer. About 10 ng of each sample (determined by the Cubit reading) is loaded in each lane. Shown is a standard DNA Agilent Chip. All the libraries we plan to multiplex are run on the same Chip. The expected products are about 450 nts for full-length histone mRNAs, with shorter lengths for the degradation intermediates. Primer dimers from PCR2 are about 120 nts and are a common contaminant. The left lane is the provided ladder for determining library size. Lanes 2, 4, 5, and 8 excellent libraries with minimal primer dimers. Lanes 1 and 6 have some primer dimer contamination, but gave good results on sequencing. Lanes 3 and 9 have excessive amounts of primer dimer. This is generally corrected by reamplifying the cDNA. Lane 7 is a failed library, which may require starting from a new reverse transcription of the ligated RNA.

Techniques Used: Chromatin Immunoprecipitation, Multiplex Assay, Sequencing

26) Product Images from "Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection"

Article Title: Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection

Journal: Silence

doi: 10.1186/1758-907X-3-9

Anticipated results. ( A ) Bioanalyzer plot. ( B ) Agarose gel for small-scale colony sequencing. Control, a PCR reaction with no bacterial colony added.
Figure Legend Snippet: Anticipated results. ( A ) Bioanalyzer plot. ( B ) Agarose gel for small-scale colony sequencing. Control, a PCR reaction with no bacterial colony added.

Techniques Used: Agarose Gel Electrophoresis, Sequencing, Polymerase Chain Reaction

27) Product Images from "Generation of Genetically Modified Mice using the CRISPR-Cas9 Genome-Editing System"

Article Title: Generation of Genetically Modified Mice using the CRISPR-Cas9 Genome-Editing System

Journal: Cold Spring Harbor protocols

doi: 10.1101/pdb.prot090704

Quality control of Cas9 mRNA in a 2100 Bioanalyzer and ssDNA donor oligo design A . Electrophoresis of in vitro transcribed Cas9 mRNA prior poly-polyadenylation and post poly-adenylation. B . Electropherogram of in vitro transcribed Cas9 mRNA prior poly-polyadenylation and post poly-adenylation. C . Representation of a sequence that can be targeted with a specific sgRNA. Insertion of EcoRI (as an example) adjacent to the PAM sequence will preclude the Cas9 nuclease from cutting again after the genome has been repaired by HDR.
Figure Legend Snippet: Quality control of Cas9 mRNA in a 2100 Bioanalyzer and ssDNA donor oligo design A . Electrophoresis of in vitro transcribed Cas9 mRNA prior poly-polyadenylation and post poly-adenylation. B . Electropherogram of in vitro transcribed Cas9 mRNA prior poly-polyadenylation and post poly-adenylation. C . Representation of a sequence that can be targeted with a specific sgRNA. Insertion of EcoRI (as an example) adjacent to the PAM sequence will preclude the Cas9 nuclease from cutting again after the genome has been repaired by HDR.

Techniques Used: Electrophoresis, In Vitro, Sequencing

28) Product Images from "Preparation of RNA 3’ End Sequencing Libraries of Total and 4-thiouracil Labeled RNA for Simultaneous Measurement of Transcription, RNA Synthesis and Decay in S. cerevisiae"

Article Title: Preparation of RNA 3’ End Sequencing Libraries of Total and 4-thiouracil Labeled RNA for Simultaneous Measurement of Transcription, RNA Synthesis and Decay in S. cerevisiae

Journal: Bio-protocol

doi: 10.21769/BioProtoc.3189

Example of Bioanalyzer traces of final sequencing libraries. Libraries shown are from samples that were not subjected to E-PAP treatment. Lanes 1-4 are from total RNA and lanes 5-11 from 10’ 4tU IP samples. All samples were successfully used for Illumina sequencing.
Figure Legend Snippet: Example of Bioanalyzer traces of final sequencing libraries. Libraries shown are from samples that were not subjected to E-PAP treatment. Lanes 1-4 are from total RNA and lanes 5-11 from 10’ 4tU IP samples. All samples were successfully used for Illumina sequencing.

Techniques Used: Sequencing

29) Product Images from "Transcriptome Profiling of Archived Sectioned Formalin-Fixed Paraffin-Embedded (AS-FFPE) Tissue for Disease Classification"

Article Title: Transcriptome Profiling of Archived Sectioned Formalin-Fixed Paraffin-Embedded (AS-FFPE) Tissue for Disease Classification

Journal: PLoS ONE

doi: 10.1371/journal.pone.0086961

Bioanalyzer results for total RNA isolated from (A) AS-FFPE HCC tissues, and (B) good, intermediate, and poor quality FC-FFPE tissues. The AS- and FC-FFPE RNA samples were extracted from different samples. AS: archived section, FFPE: formalin-fixed paraffin-embedded, HCC: hepatocellular carcinoma, FC: freshly cut, RIN: RNA Integrity Number.
Figure Legend Snippet: Bioanalyzer results for total RNA isolated from (A) AS-FFPE HCC tissues, and (B) good, intermediate, and poor quality FC-FFPE tissues. The AS- and FC-FFPE RNA samples were extracted from different samples. AS: archived section, FFPE: formalin-fixed paraffin-embedded, HCC: hepatocellular carcinoma, FC: freshly cut, RIN: RNA Integrity Number.

Techniques Used: Isolation, Formalin-fixed Paraffin-Embedded

30) Product Images from "Bias in recent miRBase annotations potentially associated with RNA quality issues"

Article Title: Bias in recent miRBase annotations potentially associated with RNA quality issues

Journal: Scientific Reports

doi: 10.1038/s41598-017-05070-0

Experimental design. ( a ) Liver, heart and brain of male mice were harvested immediately after death, divided into 8 parts of about equal size, and stored at either 4 °C or at room temperature (RT) for the indicated time periods before RNA isolation. Experiments were performed in biological triplicates. RNA integrity was measured with Bioanalyzer. Gel-like image of brain tissue is given as example. MiRNA expression profiles of one replicate were measured using microarrays. ( b ) Liver tissue of 3 male mice was harvested immediately after death and divided into 5 parts of about equal size. Three parts were immediately transferred into RNAlater (0 h), two parts were stored for 96 h at room temperature (96 h). Two samples (0 h and 96 h) were isolated using standard procedure with miRNeasy Kit without DNase digestion. Two samples (0 h and 96 h) were isolated with optional DNase digestion to exclude DNA background. From the remaining undegraded sample (0 h), total RNA without small RNAs was isolated using RNeasy Kit with optional DNase digestion. Isolated RNA was further treated with 0 U, 0.026 U and 0.67 U RNase for 30 min to generate artificial RNA degradation. RNA integrity was measured with Bioanalyzer. MiRNA expression profiles of all replicates were measured using microarrays. The schematic drawings were prepared using the Biomedical-PPT-Toolkit-Suite from Motifolio Inc., USA.
Figure Legend Snippet: Experimental design. ( a ) Liver, heart and brain of male mice were harvested immediately after death, divided into 8 parts of about equal size, and stored at either 4 °C or at room temperature (RT) for the indicated time periods before RNA isolation. Experiments were performed in biological triplicates. RNA integrity was measured with Bioanalyzer. Gel-like image of brain tissue is given as example. MiRNA expression profiles of one replicate were measured using microarrays. ( b ) Liver tissue of 3 male mice was harvested immediately after death and divided into 5 parts of about equal size. Three parts were immediately transferred into RNAlater (0 h), two parts were stored for 96 h at room temperature (96 h). Two samples (0 h and 96 h) were isolated using standard procedure with miRNeasy Kit without DNase digestion. Two samples (0 h and 96 h) were isolated with optional DNase digestion to exclude DNA background. From the remaining undegraded sample (0 h), total RNA without small RNAs was isolated using RNeasy Kit with optional DNase digestion. Isolated RNA was further treated with 0 U, 0.026 U and 0.67 U RNase for 30 min to generate artificial RNA degradation. RNA integrity was measured with Bioanalyzer. MiRNA expression profiles of all replicates were measured using microarrays. The schematic drawings were prepared using the Biomedical-PPT-Toolkit-Suite from Motifolio Inc., USA.

Techniques Used: Mouse Assay, Isolation, Expressing

31) Product Images from "The histone variant H3.3 G34W substitution in giant cell tumor of the bone link chromatin and RNA processing"

Article Title: The histone variant H3.3 G34W substitution in giant cell tumor of the bone link chromatin and RNA processing

Journal: Scientific Reports

doi: 10.1038/s41598-017-13887-y

RNA-sequencing analysis of three H3.3 WT and three H3.3 G34W primary cell lines show distinct splicing aberrations. ( a ) Electropherogram of MNase assay estimating chromatin compaction of mutated and unmutated primary cells. Plot based on 60 min incubation with MNase of H3.3 WT (blue line) and H3.3 G34W (red line) primary cell lines and BioAnalyzer data collection. ( b ) RNA splicing events of H3.3 G34W vs. H3.3 WT primary cells, extracted by the rMATS and SpliceR algorithms. The increased splicing events in H3.3 G34W when compared to H3.3 WT are given in [%]. ( c ) Plot of the significant increase of potentially novel isoforms. The data is based on the Cufflinks category j that calls for potentially novel isoforms (fragments) with at least one splice junction shared with a reference transcript. ( d ) Three examples (AURKA, NASP, and TPM2) of exon inclusion events in H3.3 G34W , first observed in RNA-seq. and here validated with the nCounter RNA-based hybridization technology. ( e ) Exon skipping in TUFT1 occur in H3.3 G34W , and probe detects junction of flanking exons to the skipped exon. ( f ) Alternative transcriptional start sites at the DDX10 and TPM1 locus. Increased read counts in their first or second introns indicated by red boxes. Plot of nCounter validations are shown in the right panel, where loss of normal start correlates with gain of alternative start.
Figure Legend Snippet: RNA-sequencing analysis of three H3.3 WT and three H3.3 G34W primary cell lines show distinct splicing aberrations. ( a ) Electropherogram of MNase assay estimating chromatin compaction of mutated and unmutated primary cells. Plot based on 60 min incubation with MNase of H3.3 WT (blue line) and H3.3 G34W (red line) primary cell lines and BioAnalyzer data collection. ( b ) RNA splicing events of H3.3 G34W vs. H3.3 WT primary cells, extracted by the rMATS and SpliceR algorithms. The increased splicing events in H3.3 G34W when compared to H3.3 WT are given in [%]. ( c ) Plot of the significant increase of potentially novel isoforms. The data is based on the Cufflinks category j that calls for potentially novel isoforms (fragments) with at least one splice junction shared with a reference transcript. ( d ) Three examples (AURKA, NASP, and TPM2) of exon inclusion events in H3.3 G34W , first observed in RNA-seq. and here validated with the nCounter RNA-based hybridization technology. ( e ) Exon skipping in TUFT1 occur in H3.3 G34W , and probe detects junction of flanking exons to the skipped exon. ( f ) Alternative transcriptional start sites at the DDX10 and TPM1 locus. Increased read counts in their first or second introns indicated by red boxes. Plot of nCounter validations are shown in the right panel, where loss of normal start correlates with gain of alternative start.

Techniques Used: RNA Sequencing Assay, Incubation, Hybridization

32) Product Images from "Epigenetic Analysis of Laser Capture Microdissected Fetal Epithelia"

Article Title: Epigenetic Analysis of Laser Capture Microdissected Fetal Epithelia

Journal: Analytical biochemistry

doi: 10.1016/j.ab.2013.07.029

Bioanalyzer scans for LCM-procured MEE RNAs using the RNAqueous ® Micro kit
Figure Legend Snippet: Bioanalyzer scans for LCM-procured MEE RNAs using the RNAqueous ® Micro kit

Techniques Used: Laser Capture Microdissection

33) Product Images from "The stage-specific testicular germ cell apoptotic response to low dose X-irradiation and 2,5-hexanedione combined exposure. I. Validation of the laser capture microdissection method for qRT-PCR array application"

Article Title: The stage-specific testicular germ cell apoptotic response to low dose X-irradiation and 2,5-hexanedione combined exposure. I. Validation of the laser capture microdissection method for qRT-PCR array application

Journal: Toxicologic pathology

doi: 10.1177/0192623314526319

LCM-derived seminiferous tubule RNA quality assessment. Digital gel (A) and electropherogram results (B–C) obtained with the Agilent 2100 Bioanalyzer. Electropherogram results are shown for before (B) and after (C) DNase treatment and RNA concentration,
Figure Legend Snippet: LCM-derived seminiferous tubule RNA quality assessment. Digital gel (A) and electropherogram results (B–C) obtained with the Agilent 2100 Bioanalyzer. Electropherogram results are shown for before (B) and after (C) DNase treatment and RNA concentration,

Techniques Used: Laser Capture Microdissection, Derivative Assay, Concentration Assay

34) Product Images from "Use of Illumina Deep Sequencing Technology To Differentiate Hepatitis C Virus Variants"

Article Title: Use of Illumina Deep Sequencing Technology To Differentiate Hepatitis C Virus Variants

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.05715-11

Evaluation of the quality of libraries. (A) The library was well refined for patient 7, but the primer and adaptor dimers were mixed in the libraries of patients 1 and 9, obtained using Bioanalyzer (Agilent). The horizontal axis shows the DNA size, and
Figure Legend Snippet: Evaluation of the quality of libraries. (A) The library was well refined for patient 7, but the primer and adaptor dimers were mixed in the libraries of patients 1 and 9, obtained using Bioanalyzer (Agilent). The horizontal axis shows the DNA size, and

Techniques Used:

35) Product Images from "Development of a fluorescent reporter system for monitoring ER stress in Chinese hamster ovary cells and its application for therapeutic protein production"

Article Title: Development of a fluorescent reporter system for monitoring ER stress in Chinese hamster ovary cells and its application for therapeutic protein production

Journal: PLoS ONE

doi: 10.1371/journal.pone.0183694

Expression analysis of UPR pathway proteins and monitoring with the RXG reporter in IgG-producing cell lines. (A) Splicing of the RXG reporter (sRXG) was determined by RTPCR followed by analysis using DNA 7500 chips with an Agilent Bioanalyzer. (B) Detection of key proteins involved in UPR by Western blot analysis. IRE1 pathway proteins XBP1 (uXBP1) and its spliced form (sXBP1), chaperones GRP94, BiP, PDI, P58 IPK , and PERK pathway proteins native PERK and eIF2α and their phosphorylated forms (p-PERK and p-eIF2α) were detected by Western blot analysis. GAPDH was used as a loading control. (C) Kinetics of QP and IVCC in IgG expressing cells during fed-batch culture. Threshold metrics for QP and IVCC critical for sustained inhibition of p-PERK are marked by dashed arrowed lines. (D) HC and LC mRNA expression of multiple RXG expressing stable IgG cell lines relative to GAPDH determined by qRTPCR. (E) Kinetics of ATF4 and CHOP mRNA expression during fed-batch culture of IgG expressing cells normalized on day 0 and expressed relative to endogenous GAPDH determined by real-time Q RTPCR. (F) Kinetics of ERAD and ERQC pathway proteins such as HERPUD1, (G) EDEM1 and Derlin1determined by real-time QRTPCR during fed-batch culture of IgG expressing cells.
Figure Legend Snippet: Expression analysis of UPR pathway proteins and monitoring with the RXG reporter in IgG-producing cell lines. (A) Splicing of the RXG reporter (sRXG) was determined by RTPCR followed by analysis using DNA 7500 chips with an Agilent Bioanalyzer. (B) Detection of key proteins involved in UPR by Western blot analysis. IRE1 pathway proteins XBP1 (uXBP1) and its spliced form (sXBP1), chaperones GRP94, BiP, PDI, P58 IPK , and PERK pathway proteins native PERK and eIF2α and their phosphorylated forms (p-PERK and p-eIF2α) were detected by Western blot analysis. GAPDH was used as a loading control. (C) Kinetics of QP and IVCC in IgG expressing cells during fed-batch culture. Threshold metrics for QP and IVCC critical for sustained inhibition of p-PERK are marked by dashed arrowed lines. (D) HC and LC mRNA expression of multiple RXG expressing stable IgG cell lines relative to GAPDH determined by qRTPCR. (E) Kinetics of ATF4 and CHOP mRNA expression during fed-batch culture of IgG expressing cells normalized on day 0 and expressed relative to endogenous GAPDH determined by real-time Q RTPCR. (F) Kinetics of ERAD and ERQC pathway proteins such as HERPUD1, (G) EDEM1 and Derlin1determined by real-time QRTPCR during fed-batch culture of IgG expressing cells.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Inhibition

36) Product Images from "An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries"

Article Title: An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries

Journal: Scientific Reports

doi: 10.1038/srep37137

Quantitative evaluation of libraries prepared from oral gingival tissue. The quality of the libraries generated from gingival tissue was assessed after in vitro transcription and again after final cDNA synthesis. ( a ) Bioanalyzer (Agilent) trace showing the fragment size distributions for the six libraries after amplification by in vitro transcription. The traces from the automated protocol are shown in red while the traces from the manual protocol are shown in blue. The traces were also used to estimate the concentrations of the samples, which are illustrated in the left part of the boxplot ( c ). After the final cDNA synthesis the libraries were evaluated by quantitative PCR (qPCR) ( b ). The black vertical line marks the signal threshold at which point the cycle threshold (Ct) values were noted. The Ct values for the libraries as obtained from the qPCR are plotted in the right part of the boxplot ( c ).
Figure Legend Snippet: Quantitative evaluation of libraries prepared from oral gingival tissue. The quality of the libraries generated from gingival tissue was assessed after in vitro transcription and again after final cDNA synthesis. ( a ) Bioanalyzer (Agilent) trace showing the fragment size distributions for the six libraries after amplification by in vitro transcription. The traces from the automated protocol are shown in red while the traces from the manual protocol are shown in blue. The traces were also used to estimate the concentrations of the samples, which are illustrated in the left part of the boxplot ( c ). After the final cDNA synthesis the libraries were evaluated by quantitative PCR (qPCR) ( b ). The black vertical line marks the signal threshold at which point the cycle threshold (Ct) values were noted. The Ct values for the libraries as obtained from the qPCR are plotted in the right part of the boxplot ( c ).

Techniques Used: Generated, In Vitro, Amplification, Real-time Polymerase Chain Reaction

Quantitative evaluation of libraries prepared from total reference RNA. Sample-to-sample variation was investigated by small red triangles assessing the libraries at two points during the library preparation process. ( a ) The first evaluation is performed after in vitro transcription and checks the library concentrations and fragment lengths using a Bioanalyzer (Agilent). ( b ) After reverse transcription, a quantitative PCR (qPCR) was carried out to determine the suitable number of PCR cycles when indexing the finished libraries. The black vertical line marks the signal threshold at which point the cycle threshold (Ct) values were obtained. The spread of Ct values is illustrated in the boxplot ( c ), which also shows the variation in sample concentration as measured by the Bioanalyzer after in vitro transcription.
Figure Legend Snippet: Quantitative evaluation of libraries prepared from total reference RNA. Sample-to-sample variation was investigated by small red triangles assessing the libraries at two points during the library preparation process. ( a ) The first evaluation is performed after in vitro transcription and checks the library concentrations and fragment lengths using a Bioanalyzer (Agilent). ( b ) After reverse transcription, a quantitative PCR (qPCR) was carried out to determine the suitable number of PCR cycles when indexing the finished libraries. The black vertical line marks the signal threshold at which point the cycle threshold (Ct) values were obtained. The spread of Ct values is illustrated in the boxplot ( c ), which also shows the variation in sample concentration as measured by the Bioanalyzer after in vitro transcription.

Techniques Used: In Vitro, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Concentration Assay

37) Product Images from "Early dynamics of photosynthetic Lhcf2 and Lhcf15 transcription and mRNA stabilities in response to herbivory-related decadienal in Phaeodactylum tricornutum"

Article Title: Early dynamics of photosynthetic Lhcf2 and Lhcf15 transcription and mRNA stabilities in response to herbivory-related decadienal in Phaeodactylum tricornutum

Journal: Scientific Reports

doi: 10.1038/s41598-020-58885-9

In vivo incorporation of 4-TU into RNA of P. tricornutum . ( a ) Schematic workflow of 4-TU labeling into RNA and detection. Bioanalyzer profiles of RNA fractions from cell cultures treated with 0.5 mM 4-TU ( b ) or 0.1% DMSO as solvent control ( c ) for 1.5 h. From each treated culture, total RNA represents the biotinylated RNA fraction and eluted RNA represents the newly synthesized RNA fraction. Equal masses of biotinylated RNA fractions were used to prepare the eluted RNA fractions.
Figure Legend Snippet: In vivo incorporation of 4-TU into RNA of P. tricornutum . ( a ) Schematic workflow of 4-TU labeling into RNA and detection. Bioanalyzer profiles of RNA fractions from cell cultures treated with 0.5 mM 4-TU ( b ) or 0.1% DMSO as solvent control ( c ) for 1.5 h. From each treated culture, total RNA represents the biotinylated RNA fraction and eluted RNA represents the newly synthesized RNA fraction. Equal masses of biotinylated RNA fractions were used to prepare the eluted RNA fractions.

Techniques Used: In Vivo, Labeling, Synthesized

38) Product Images from "High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics"

Article Title: High-throughput single-cell DNA sequencing of acute myeloid leukemia tumors with droplet microfluidics

Journal: Genome Research

doi: 10.1101/gr.232272.117

Protease-based workflows provide improved genomic DNA amplification. ( A ) When protease enzyme is left out of the workflow for single-cell gDNA PCR in droplets, only ∼5% of DU145 cells (viability stained on the x -axis) are positive for SRY TaqMan reaction fluorescence ( y -axis). Using protease during cell lysis improves the DU145 cell detection rate to ∼98% (red points in upper right quadrant). Points in the plot represent droplets. ( B ) Bioanalyzer traces of sequencing libraries prepared from cells processed through the workflow with (black trace) or without (red trace) the use of protease indicate that PCR amplification in droplets is improved with proteolysis. The two-step workflow with protease enables better sequencing coverage depth per cell across the eight amplified target loci listed on the x -axis ( C ).
Figure Legend Snippet: Protease-based workflows provide improved genomic DNA amplification. ( A ) When protease enzyme is left out of the workflow for single-cell gDNA PCR in droplets, only ∼5% of DU145 cells (viability stained on the x -axis) are positive for SRY TaqMan reaction fluorescence ( y -axis). Using protease during cell lysis improves the DU145 cell detection rate to ∼98% (red points in upper right quadrant). Points in the plot represent droplets. ( B ) Bioanalyzer traces of sequencing libraries prepared from cells processed through the workflow with (black trace) or without (red trace) the use of protease indicate that PCR amplification in droplets is improved with proteolysis. The two-step workflow with protease enables better sequencing coverage depth per cell across the eight amplified target loci listed on the x -axis ( C ).

Techniques Used: Amplification, Polymerase Chain Reaction, Staining, Fluorescence, Lysis, Sequencing

39) Product Images from "Evaluation of Candidate Reference Genes for Real-Time Quantitative PCR of Plant Samples Using Purified cDNA as Template"

Article Title: Evaluation of Candidate Reference Genes for Real-Time Quantitative PCR of Plant Samples Using Purified cDNA as Template

Journal: Plant Molecular Biology Reporter / Ispmb

doi: 10.1007/s11105-008-0072-1

Total RNA profile of plant tissues as determined by microcapillary electrophoresis can vary significantly depending on growth conditions, but purified cDNA profiles are similar. a Total RNA electropherogram from whole, light-grown Arabidopsis seedlings (the two large cytosolic rRNA fragments and the 16S plastidic rRNA fragments are indicated). b Purified cDNA following reverse transcription of a . c Total RNA from whole, dark-grown (etiolated) Arabidopsis seedlings. d Purified cDNA following reverse transcription of c . Samples in a and c were analyzed on an Agilent Bioanalyzer using an RNA Nano LabChip®. Samples in b and d were analyzed on an RNA Pico LabChip®
Figure Legend Snippet: Total RNA profile of plant tissues as determined by microcapillary electrophoresis can vary significantly depending on growth conditions, but purified cDNA profiles are similar. a Total RNA electropherogram from whole, light-grown Arabidopsis seedlings (the two large cytosolic rRNA fragments and the 16S plastidic rRNA fragments are indicated). b Purified cDNA following reverse transcription of a . c Total RNA from whole, dark-grown (etiolated) Arabidopsis seedlings. d Purified cDNA following reverse transcription of c . Samples in a and c were analyzed on an Agilent Bioanalyzer using an RNA Nano LabChip®. Samples in b and d were analyzed on an RNA Pico LabChip®

Techniques Used: Electrophoresis, Purification

40) Product Images from "Comparison of DNA Quantification Methods for Next Generation Sequencing"

Article Title: Comparison of DNA Quantification Methods for Next Generation Sequencing

Journal: Scientific Reports

doi: 10.1038/srep24067

Comparison of four titration techniques for NGS. ( a ) Diagram of the Next generation sequencing (NGS) experimental design. Using four quantification methods, we titrated DNA libraries prepared from HeLa cells, following manufacturers’ instructions. Six different indexes were added at the amplification step. Of the eight lanes on the NGS flowcell, four lanes were used to compare each method (unique index lane) and four lanes for pooling accuracy (pool of six indexes per lane). ( b ) Bioanalyzer image of the libraries, showing a homogenous smear of DNA from 280 to 450 bp. All six indexes show similarly average sizes. ( c ) Result of the quantification for all indexes using QuBit, qPCR, ddPCR and ddPCR-Tail approaches, respectively. All quantifications (except ddPCR-Tail) were done following manufacturer instructions (Invitrogen, BioRad, and KapaBiosystem). The ddPCR-Tail strategy used the same apparatus as the ddPCR with slight modifications (50 nM primers; three-step PCR-annealing at 65 °C for 30 seconds, extension time of 30 seconds at 72 °C). Experiments done in triplicate, mean calculated using dilution curve (average of 12 values per quantification, 6 for QuBit). Mean ± SD shown.
Figure Legend Snippet: Comparison of four titration techniques for NGS. ( a ) Diagram of the Next generation sequencing (NGS) experimental design. Using four quantification methods, we titrated DNA libraries prepared from HeLa cells, following manufacturers’ instructions. Six different indexes were added at the amplification step. Of the eight lanes on the NGS flowcell, four lanes were used to compare each method (unique index lane) and four lanes for pooling accuracy (pool of six indexes per lane). ( b ) Bioanalyzer image of the libraries, showing a homogenous smear of DNA from 280 to 450 bp. All six indexes show similarly average sizes. ( c ) Result of the quantification for all indexes using QuBit, qPCR, ddPCR and ddPCR-Tail approaches, respectively. All quantifications (except ddPCR-Tail) were done following manufacturer instructions (Invitrogen, BioRad, and KapaBiosystem). The ddPCR-Tail strategy used the same apparatus as the ddPCR with slight modifications (50 nM primers; three-step PCR-annealing at 65 °C for 30 seconds, extension time of 30 seconds at 72 °C). Experiments done in triplicate, mean calculated using dilution curve (average of 12 values per quantification, 6 for QuBit). Mean ± SD shown.

Techniques Used: Titration, Next-Generation Sequencing, Amplification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Titration of six NGS libraries with low abundance with qPCR and ddPCR Tail. ( a ) Comparison of two methods for low abundance NGS libraries, titration results using qPCR and the ddPCR-Tail system. Assay done in triplicate and mean calculated using dilution correction factors (average of 18 values per sample). Mean ± SD shown. ( b ) Bioanalyzer image results used to calculate final molarity for the qPCR measurement, algorithm provided by KapaBiosystem using the average size of the NGS library combined with the qPCR standard curve. ( c ) All libraries were successfully quantified with high confidence (linear regression all in 0.9), ddPCR Tail strategy showed a better overall linearity regardless of the heterogeneity of the libraries.
Figure Legend Snippet: Titration of six NGS libraries with low abundance with qPCR and ddPCR Tail. ( a ) Comparison of two methods for low abundance NGS libraries, titration results using qPCR and the ddPCR-Tail system. Assay done in triplicate and mean calculated using dilution correction factors (average of 18 values per sample). Mean ± SD shown. ( b ) Bioanalyzer image results used to calculate final molarity for the qPCR measurement, algorithm provided by KapaBiosystem using the average size of the NGS library combined with the qPCR standard curve. ( c ) All libraries were successfully quantified with high confidence (linear regression all in 0.9), ddPCR Tail strategy showed a better overall linearity regardless of the heterogeneity of the libraries.

Techniques Used: Titration, Next-Generation Sequencing, Real-time Polymerase Chain Reaction

Related Articles

SYBR Green Assay:

Article Title: Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes
Article Snippet: .. Materials and equipment Purified Nextera libraries from Module 4 High Sensitivity DNA kit for BioAnalyzer (Agilent 5067–4626) TE buffer 50mL reagent reservoirs 96-well plate with flat transparent bottom for fluorometry (e.g. Corning 3603) SYBR Green I (Life technologies S-7563) DNA standards in range of 1-10ng/μl (we use those that come with Life Q-33120) Plate reader with SYBR-compatible filters BioAnalyzer (Agilent 2100) or similar DNA fragment-size assay system. .. Procedure Perform steps 1–7 of Module 1 to quantify DNA concentration across all samples.

Concentration Assay:

Article Title: Identification of N6,N6-Dimethyladenosine in Transfer RNA from Mycobacterium bovis Bacille Calmette-Guérin
Article Snippet: .. The quality and concentration of the resulting small RNA mixture was assessed with a Bioanalyzer (Agilent Small RNA Kit), with tRNA comprising > 95% of the small RNA species present in the mixture, with no detectable 5S rRNA ( ). .. In one study, tRNA was purified from BCG small RNA isolates by size-exclusion HPLC using an Agilent SEC3 300 Å, 7.8 × 300 mm column eluted with 10 mM ammonium acetate.

Purification:

Article Title: Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes
Article Snippet: .. Materials and equipment Purified Nextera libraries from Module 4 High Sensitivity DNA kit for BioAnalyzer (Agilent 5067–4626) TE buffer 50mL reagent reservoirs 96-well plate with flat transparent bottom for fluorometry (e.g. Corning 3603) SYBR Green I (Life technologies S-7563) DNA standards in range of 1-10ng/μl (we use those that come with Life Q-33120) Plate reader with SYBR-compatible filters BioAnalyzer (Agilent 2100) or similar DNA fragment-size assay system. .. Procedure Perform steps 1–7 of Module 1 to quantify DNA concentration across all samples.

Article Title: Exploring genome wide bisulfite sequencing for DNA methylation analysis in livestock: a technical assessment
Article Snippet: .. All PCR reactions for RRBS and WGBS were purified using Clean and concentratorTM -5 column (Zymo, Irvine, CA, USA), analyzed on a bioanalyzer (Agilent, Santa Clara, CA, USA) and each library was sequenced on one lane of an Illumina HiSeq sequencer using 100 bp paired-end reads (National Centre for Genome Resources, Santa Fe, NM, USA). ..

Isolation:

Article Title: Horizontal transfer of exosomal microRNAs transduce apoptotic signals between pancreatic beta-cells
Article Snippet: .. The size of the RNAs isolated from MIN6B1 cells or present in exosomes was analyzed using a Bioanalyzer (Agilent Technology). .. Quantification of mature miRNA levels Expression of mature miRNAs was quantified with the miRCURY LNATM Universal RT microRNA PCR kit (Exiqon).

Polymerase Chain Reaction:

Article Title: Exploring genome wide bisulfite sequencing for DNA methylation analysis in livestock: a technical assessment
Article Snippet: .. All PCR reactions for RRBS and WGBS were purified using Clean and concentratorTM -5 column (Zymo, Irvine, CA, USA), analyzed on a bioanalyzer (Agilent, Santa Clara, CA, USA) and each library was sequenced on one lane of an Illumina HiSeq sequencer using 100 bp paired-end reads (National Centre for Genome Resources, Santa Fe, NM, USA). ..

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    Agilent technologies agilent bioanalyzer
    Urinary microvesicle RNA integrity and alignment to the genome. a) RNA isolated from urinary microvesicles was shown to be of high integrity with prominent 18S and 28S rRNA peaks when analyzed using the <t>Agilent</t> <t>Bioanalyzer.</t> Red trace 1.7 ng RNA with DNase, Blue trace 2.2 ng RNA without DNase. b) Flow chart outlining sample processing. An initial RNase and DNase digestion was carried out to remove extraneous nucleic acids co-isolating with the microvesicle pellet. To determine the proportion of potential DNA inside the microvesicles the extracted RNA was divided into two groups; No DNase digestion (-DNase), which yields RNA+DNA and DNase digested (+DNase) which yields RNA. c) Both the -DNase and the +DNase samples showed a similar trend in read distribution with ∼88% of reads mapping to rRNA, ∼4% mapping to genes and ∼6% mapping to ncRNA. A smaller proportion of reads (
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    Total RNA concentrations from different number of renal cells. Total RNA concentrations were measured using <t>Agilent</t> 2100 <t>Bioanalyzer</t> 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.
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    Urinary microvesicle RNA integrity and alignment to the genome. a) RNA isolated from urinary microvesicles was shown to be of high integrity with prominent 18S and 28S rRNA peaks when analyzed using the Agilent Bioanalyzer. Red trace 1.7 ng RNA with DNase, Blue trace 2.2 ng RNA without DNase. b) Flow chart outlining sample processing. An initial RNase and DNase digestion was carried out to remove extraneous nucleic acids co-isolating with the microvesicle pellet. To determine the proportion of potential DNA inside the microvesicles the extracted RNA was divided into two groups; No DNase digestion (-DNase), which yields RNA+DNA and DNase digested (+DNase) which yields RNA. c) Both the -DNase and the +DNase samples showed a similar trend in read distribution with ∼88% of reads mapping to rRNA, ∼4% mapping to genes and ∼6% mapping to ncRNA. A smaller proportion of reads (

    Journal: PLoS ONE

    Article Title: Massively Parallel Sequencing of Human Urinary Exosome/Microvesicle RNA Reveals a Predominance of Non-Coding RNA

    doi: 10.1371/journal.pone.0096094

    Figure Lengend Snippet: Urinary microvesicle RNA integrity and alignment to the genome. a) RNA isolated from urinary microvesicles was shown to be of high integrity with prominent 18S and 28S rRNA peaks when analyzed using the Agilent Bioanalyzer. Red trace 1.7 ng RNA with DNase, Blue trace 2.2 ng RNA without DNase. b) Flow chart outlining sample processing. An initial RNase and DNase digestion was carried out to remove extraneous nucleic acids co-isolating with the microvesicle pellet. To determine the proportion of potential DNA inside the microvesicles the extracted RNA was divided into two groups; No DNase digestion (-DNase), which yields RNA+DNA and DNase digested (+DNase) which yields RNA. c) Both the -DNase and the +DNase samples showed a similar trend in read distribution with ∼88% of reads mapping to rRNA, ∼4% mapping to genes and ∼6% mapping to ncRNA. A smaller proportion of reads (

    Article Snippet: Isolated RNA was analyzed on a RNA Pico 6000 chip (Agilent, CA) using an Agilent Bioanalyzer to check for integrity.

    Techniques: Isolation, Flow Cytometry

    VELCRO-IP RT-qPCR serves as a proof-of-principle to identify novel hES9S-interacting 5’ UTRs and mRNA fragmentation. Related to Figure 3. (A) Schematic of in vitro transcripts used for the proof-of-principle experiment of the VELCRO-IP RT-qPCR. Reproduced from Figure 3B . (B) For qualitative analysis of the integrity of in vitro transcripts, RNAs were subjected to 4-20% polyacrylamide/TBE/native PAGE and visualized by SYBR Gold staining. (C) Analysis of total RNA in the 3xFlag peptide elution by RT-qPCR using same volumes of RNA per sample for the RT. Normalization of Ct values for Fluc to the 18S rRNA tag internally controls for ribosome-IP efficiency per sample. The native/WT sample was used to normalize for fold enrichment of RNA binding (set to 1). Representation of the raw data in Figure 3D . Average RNA fold enrichment, SEM, n = 5; ns, not significant. (D) Full view of the Bioanalyzer (Agilent) quantification and electronic gel analysis in Figure 3G, H is shown for optimization of mouse mRNA fragmentation from C3H/10T1/2 cells and stage E11.5 mouse embryos. The marker (M, grey) is overlaid for reference. (E) Full view of the Bioanalyzer (Agilent) quantification and electronic gel analysis in Figure 4B is shown for the eluted and yeast rRNA-depleted mouse embryo RNA from three independent replicates of WT and hES9S VELCRO-IP experiments. The marker (M, grey) is overlaid for reference.

    Journal: bioRxiv

    Article Title: VELCRO-IP RNA-seq explores ribosome expansion segment function in translation genome-wide

    doi: 10.1101/2020.07.01.179515

    Figure Lengend Snippet: VELCRO-IP RT-qPCR serves as a proof-of-principle to identify novel hES9S-interacting 5’ UTRs and mRNA fragmentation. Related to Figure 3. (A) Schematic of in vitro transcripts used for the proof-of-principle experiment of the VELCRO-IP RT-qPCR. Reproduced from Figure 3B . (B) For qualitative analysis of the integrity of in vitro transcripts, RNAs were subjected to 4-20% polyacrylamide/TBE/native PAGE and visualized by SYBR Gold staining. (C) Analysis of total RNA in the 3xFlag peptide elution by RT-qPCR using same volumes of RNA per sample for the RT. Normalization of Ct values for Fluc to the 18S rRNA tag internally controls for ribosome-IP efficiency per sample. The native/WT sample was used to normalize for fold enrichment of RNA binding (set to 1). Representation of the raw data in Figure 3D . Average RNA fold enrichment, SEM, n = 5; ns, not significant. (D) Full view of the Bioanalyzer (Agilent) quantification and electronic gel analysis in Figure 3G, H is shown for optimization of mouse mRNA fragmentation from C3H/10T1/2 cells and stage E11.5 mouse embryos. The marker (M, grey) is overlaid for reference. (E) Full view of the Bioanalyzer (Agilent) quantification and electronic gel analysis in Figure 4B is shown for the eluted and yeast rRNA-depleted mouse embryo RNA from three independent replicates of WT and hES9S VELCRO-IP experiments. The marker (M, grey) is overlaid for reference.

    Article Snippet: DNA fragments were purified for Illumina sequencing, subjected to analysis using the High Sensitivity DNA Assay (Agilent) on a Bioanalyzer (Agilent) and all DNA libraries were pooled to a final concentration of 4 nM.

    Techniques: Quantitative RT-PCR, In Vitro, Clear Native PAGE, Staining, RNA Binding Assay, Marker

    VELCRO-IP RNA-seq identifies global ES-mRNA interactions with positional resolution on mRNAs. (A) For VELCRO-IP RNA-seq, mRNA was isolated from stage E11.5 mouse embryos, fragmented to 100-200 nt and used as input for the IP. Total RNA obtained from all samples after elution and yeast rRNA depletion obtains ribosome-bound mouse mRNA that was subjected to library preparation and Illumina high-throughput sequencing (NextSeq). We included an mRNA fragment input sample for reference. The distribution of mRNA fragment lengths for all sequenced libraries is plotted and the median fragment length is 246 nt. We then map all reads to the mouse and yeast transcriptomes and only further analyze reads exclusively mapping to mouse mRNAs. (B) Eluted and yeast rRNA-depleted mouse RNA from three independent replicates of WT and hES9S VELCRO-IP experiments were analyzed on a mRNA Pico Chip (Agilent) on a Bioanalyzer (Agilent) and a zoomed-in view of the Bioanalyzer quantification and electronic gel analysis is shown. The marker (M, grey) is overlaid for reference. See also Figure S3E . (C) WB analysis as in Figure 3C to monitor efficient IP of 40S ribosomes for RNA-seq of mouse mRNA fragments. Representative of n = 3 is shown. (D) Kernel density of the distribution of t-statistics for test of differential enrichment of mRNA fragments bound to hES9S vs WT ribosomes is plotted in black. Empirical estimate of the decomposition of the distribution to null and non-null tests are plotted in grey and red, respectively. Dotted line indicates where the local FDR of 0.05 is. (E) RNA-seq results of independent replicates (n = 3) for each WT and hES9S samples. Normalized log read counts are presented for WT and hES9S-enriched mRNA fragments. Fragments less than FDR

    Journal: bioRxiv

    Article Title: VELCRO-IP RNA-seq explores ribosome expansion segment function in translation genome-wide

    doi: 10.1101/2020.07.01.179515

    Figure Lengend Snippet: VELCRO-IP RNA-seq identifies global ES-mRNA interactions with positional resolution on mRNAs. (A) For VELCRO-IP RNA-seq, mRNA was isolated from stage E11.5 mouse embryos, fragmented to 100-200 nt and used as input for the IP. Total RNA obtained from all samples after elution and yeast rRNA depletion obtains ribosome-bound mouse mRNA that was subjected to library preparation and Illumina high-throughput sequencing (NextSeq). We included an mRNA fragment input sample for reference. The distribution of mRNA fragment lengths for all sequenced libraries is plotted and the median fragment length is 246 nt. We then map all reads to the mouse and yeast transcriptomes and only further analyze reads exclusively mapping to mouse mRNAs. (B) Eluted and yeast rRNA-depleted mouse RNA from three independent replicates of WT and hES9S VELCRO-IP experiments were analyzed on a mRNA Pico Chip (Agilent) on a Bioanalyzer (Agilent) and a zoomed-in view of the Bioanalyzer quantification and electronic gel analysis is shown. The marker (M, grey) is overlaid for reference. See also Figure S3E . (C) WB analysis as in Figure 3C to monitor efficient IP of 40S ribosomes for RNA-seq of mouse mRNA fragments. Representative of n = 3 is shown. (D) Kernel density of the distribution of t-statistics for test of differential enrichment of mRNA fragments bound to hES9S vs WT ribosomes is plotted in black. Empirical estimate of the decomposition of the distribution to null and non-null tests are plotted in grey and red, respectively. Dotted line indicates where the local FDR of 0.05 is. (E) RNA-seq results of independent replicates (n = 3) for each WT and hES9S samples. Normalized log read counts are presented for WT and hES9S-enriched mRNA fragments. Fragments less than FDR

    Article Snippet: DNA fragments were purified for Illumina sequencing, subjected to analysis using the High Sensitivity DNA Assay (Agilent) on a Bioanalyzer (Agilent) and all DNA libraries were pooled to a final concentration of 4 nM.

    Techniques: RNA Sequencing Assay, Isolation, Next-Generation Sequencing, Chromatin Immunoprecipitation, Marker, Western Blot

    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.

    Journal: BMC Research Notes

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

    doi: 10.1186/1756-0500-7-62

    Figure Lengend 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.

    Article Snippet: RNA integrity number (RIN) RIN was measured using Agilent 2100 Bioanalyzer with Agilent RNA 6000 Pico Kit.

    Techniques: Concentration Assay, Produced

    Analyses of the various antibody formats ( A ) To assess the purity and confirm the size of each reagent, the various antibody formats were analyzed by Agilent 2100 bioanalyzer. Proteins were assessed under non-reduced (NR) and reduced conditions (R). 1, IgG; 2, heavy chain; 3, light chain; 4, F(ab)’ 2 ; 5, VH-CH1-hinge; 6, monovalent antibody; 7, hinge-Fc; 8, Fab; 9, light chain and VH-CH1. ( B ) The binding of Hu 15C1 (black circles), monovalent Hu 15C1 (gray triangles), F(ab)’ 2 Hu 15C1 (open circles) and Fab Hu 15C1 (open diamonds) to TLR4 was analyzed by competitive ELISA. To compare the different antibody formats, the same number of binding site was used, i.e., the molar concentration of monovalent and Fab is twice the molar concentration of IgG and F(ab)’ 2 . Results are normalized and expressed as mean ± SD of duplicates. An F test was used to compare the fitted curves of different groups. ns: not significant.

    Journal: mAbs

    Article Title: Maximizing the potency of an anti-TLR4 monoclonal antibody by exploiting proximity to Fcγ receptors

    doi: 10.4161/19420862.2014.975098

    Figure Lengend Snippet: Analyses of the various antibody formats ( A ) To assess the purity and confirm the size of each reagent, the various antibody formats were analyzed by Agilent 2100 bioanalyzer. Proteins were assessed under non-reduced (NR) and reduced conditions (R). 1, IgG; 2, heavy chain; 3, light chain; 4, F(ab)’ 2 ; 5, VH-CH1-hinge; 6, monovalent antibody; 7, hinge-Fc; 8, Fab; 9, light chain and VH-CH1. ( B ) The binding of Hu 15C1 (black circles), monovalent Hu 15C1 (gray triangles), F(ab)’ 2 Hu 15C1 (open circles) and Fab Hu 15C1 (open diamonds) to TLR4 was analyzed by competitive ELISA. To compare the different antibody formats, the same number of binding site was used, i.e., the molar concentration of monovalent and Fab is twice the molar concentration of IgG and F(ab)’ 2 . Results are normalized and expressed as mean ± SD of duplicates. An F test was used to compare the fitted curves of different groups. ns: not significant.

    Article Snippet: The purity and chain composition of each reagent was assessed using an Agilent 2100 bioanalyzer ( ).

    Techniques: Binding Assay, Competitive ELISA, Concentration Assay