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Agilent technologies rna 6000 pico kit
Effects of proteinase K and RNase A treatment on the relative quantity of EV-incorporated miRNAs and RNA profiles in whole plasma and EVs.  a  RT-qPCR analysis of miRNA levels in EVs treated with RNase A alone or with a combination of proteinase K and RNase A relatively to untreated EVs. Bars show the mean percentage in EVs from 3 healthy individuals.  b  A representative RNA profile from whole plasma and EVs treated with proteinase K and RNase A obtained by Bioanlyzer RNA 6000 Pico chip
Rna 6000 Pico Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Detection of circulating miRNAs: comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients"

Article Title: Detection of circulating miRNAs: comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients

Journal: BMC Cancer

doi: 10.1186/s12885-017-3737-z

Effects of proteinase K and RNase A treatment on the relative quantity of EV-incorporated miRNAs and RNA profiles in whole plasma and EVs.  a  RT-qPCR analysis of miRNA levels in EVs treated with RNase A alone or with a combination of proteinase K and RNase A relatively to untreated EVs. Bars show the mean percentage in EVs from 3 healthy individuals.  b  A representative RNA profile from whole plasma and EVs treated with proteinase K and RNase A obtained by Bioanlyzer RNA 6000 Pico chip
Figure Legend Snippet: Effects of proteinase K and RNase A treatment on the relative quantity of EV-incorporated miRNAs and RNA profiles in whole plasma and EVs. a RT-qPCR analysis of miRNA levels in EVs treated with RNase A alone or with a combination of proteinase K and RNase A relatively to untreated EVs. Bars show the mean percentage in EVs from 3 healthy individuals. b A representative RNA profile from whole plasma and EVs treated with proteinase K and RNase A obtained by Bioanlyzer RNA 6000 Pico chip

Techniques Used: Quantitative RT-PCR, Chromatin Immunoprecipitation

2) Product Images from "Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes"

Article Title: Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

Journal: Scientific Reports

doi: 10.1038/srep41114

Depletion of rRNA from RNA samples isolated from dual species  P. aeruginosa / S. aureus  cultures. A total of 2 μg of DNAse-treated RNA, isolated form a dual species  P. aeruginosa  PAO1 and  S. aureus  ATCC6538 culture, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( A ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( B ) MICROBExpress or ( C ) Ribo-Zero kits are shown. ( D ) Percent of rRNA contamination as reported by the Agilent Bioanalyzer 2100 Expert Software in RNA samples from single and dual species cultures of  P. aeruginosa  and/or  S. aureus  treated with the Ribo-Zero or MICROBExpress kits. Untreated input RNA (Total) or RNA processed via Ribo-Zero or MICROBExpress treatment was subjected to qPCR analysis of  P. aeruginosa  ( E , G ) or  S. aureus  ( F , H ) rRNA transcript abundance. ( E , F ) qPCR threshold cycle (Cq) for the indicated rRNA transcripts. ( G , H ) Copy numbers of  P. aeruginosa  or  S. aureus  16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.
Figure Legend Snippet: Depletion of rRNA from RNA samples isolated from dual species P. aeruginosa / S. aureus cultures. A total of 2 μg of DNAse-treated RNA, isolated form a dual species P. aeruginosa PAO1 and S. aureus ATCC6538 culture, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( A ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( B ) MICROBExpress or ( C ) Ribo-Zero kits are shown. ( D ) Percent of rRNA contamination as reported by the Agilent Bioanalyzer 2100 Expert Software in RNA samples from single and dual species cultures of P. aeruginosa and/or S. aureus treated with the Ribo-Zero or MICROBExpress kits. Untreated input RNA (Total) or RNA processed via Ribo-Zero or MICROBExpress treatment was subjected to qPCR analysis of P. aeruginosa ( E , G ) or S. aureus ( F , H ) rRNA transcript abundance. ( E , F ) qPCR threshold cycle (Cq) for the indicated rRNA transcripts. ( G , H ) Copy numbers of P. aeruginosa or S. aureus 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

Techniques Used: Isolation, Software, Real-time Polymerase Chain Reaction, Standard Deviation

Quantitative and qualitative comparison of RNA recovered following rRNA depletion. A total of 4 μg of DNAse-treated RNA, isolated form 3-day-old  P. aeruginosa  PAO1 biofilms, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit and the Life Technologies RiboMinus Transcriptome Isolation Kit, Bacteria. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using Qubit fluorimetric quantitation with the Qubit RNA HS Assay Kit. Yields are reported as total RNA recovered ( A ) and as percentage of the input RNA ( B ). The RNA samples were also assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( C ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( D ) MICROBExpress, ( E ) RiboMinus, or ( F ) Ribo-Zero kits are shown. Dashed lines indicate peaks corresponding to 16S and 23S RNA traces, which were detected in total RNA, MICROBExpress, and RiboMins samples. ( G ) The area of 16S and 23S rRNA peaks as percent of the total detected RNA was estimated, as determined using the 2100 Expert Software. RFU, relative fluorescence units. Experiments were repeated using three biological replicates.
Figure Legend Snippet: Quantitative and qualitative comparison of RNA recovered following rRNA depletion. A total of 4 μg of DNAse-treated RNA, isolated form 3-day-old P. aeruginosa PAO1 biofilms, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit and the Life Technologies RiboMinus Transcriptome Isolation Kit, Bacteria. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using Qubit fluorimetric quantitation with the Qubit RNA HS Assay Kit. Yields are reported as total RNA recovered ( A ) and as percentage of the input RNA ( B ). The RNA samples were also assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( C ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( D ) MICROBExpress, ( E ) RiboMinus, or ( F ) Ribo-Zero kits are shown. Dashed lines indicate peaks corresponding to 16S and 23S RNA traces, which were detected in total RNA, MICROBExpress, and RiboMins samples. ( G ) The area of 16S and 23S rRNA peaks as percent of the total detected RNA was estimated, as determined using the 2100 Expert Software. RFU, relative fluorescence units. Experiments were repeated using three biological replicates.

Techniques Used: Isolation, Quantitation Assay, RNA HS Assay, Software, Fluorescence

rRNA depletion treatments of RNA derived from planktonic  P. aeruginosa  PAO1 and  Staphylococcus aureus  ATCC6538 cells. A total of 2 μg of DNAse-treated RNA, isolated from exponential phase  P. aeruginosa  PAO1 or  Staphylococcus aureus  ATCC6538 cells, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of starting total RNA material and aliquots of the RNA samples that have been processed using the MICROBExpress or Ribo-Zero kits are shown for the  P. aeruginosa  ( A ) and  S. aureus  ( B ) samples. The samples were subsequently subjected to qPCR analysis of rRNA transcript abundance, with qPCR threshold cycle (Cq) for the indicated rRNA transcripts shown for  P. aeruginosa  ( C ) and  S. aureus  ( E ). Copy numbers of  P. aeruginosa  ( D ) and  S. aureus  ( F ) 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.
Figure Legend Snippet: rRNA depletion treatments of RNA derived from planktonic P. aeruginosa PAO1 and Staphylococcus aureus ATCC6538 cells. A total of 2 μg of DNAse-treated RNA, isolated from exponential phase P. aeruginosa PAO1 or Staphylococcus aureus ATCC6538 cells, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of starting total RNA material and aliquots of the RNA samples that have been processed using the MICROBExpress or Ribo-Zero kits are shown for the P. aeruginosa ( A ) and S. aureus ( B ) samples. The samples were subsequently subjected to qPCR analysis of rRNA transcript abundance, with qPCR threshold cycle (Cq) for the indicated rRNA transcripts shown for P. aeruginosa ( C ) and S. aureus ( E ). Copy numbers of P. aeruginosa ( D ) and S. aureus ( F ) 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

Techniques Used: Derivative Assay, Isolation, Real-time Polymerase Chain Reaction, Standard Deviation

3) Product Images from "Exosomal miRNAs from Peritoneum Lavage Fluid as Potential Prognostic Biomarkers of Peritoneal Metastasis in Gastric Cancer"

Article Title: Exosomal miRNAs from Peritoneum Lavage Fluid as Potential Prognostic Biomarkers of Peritoneal Metastasis in Gastric Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0130472

Exosomal miRNA expression in malignant ascites (MA), peritoneal lavage fluid (PLF), and cell culture media (CM). Variation and commonality between the samples were examined. Evaluation of total exosomal RNA used a Bioanalyzer 2100. The electropherograms show the size distribution (nucleotides, nt) and fluorescence intensity (FU) of total exosomal RNA isolated from CM, MA, and PLF. The lowest spike (around 25 nt) in each sample is the marker of the Agilent RNA 6000 Pico kit.
Figure Legend Snippet: Exosomal miRNA expression in malignant ascites (MA), peritoneal lavage fluid (PLF), and cell culture media (CM). Variation and commonality between the samples were examined. Evaluation of total exosomal RNA used a Bioanalyzer 2100. The electropherograms show the size distribution (nucleotides, nt) and fluorescence intensity (FU) of total exosomal RNA isolated from CM, MA, and PLF. The lowest spike (around 25 nt) in each sample is the marker of the Agilent RNA 6000 Pico kit.

Techniques Used: Expressing, Cell Culture, Fluorescence, Isolation, Marker

4) Product Images from "Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma"

Article Title: Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma

Journal: Journal of Translational Medicine

doi: 10.1186/s12967-017-1374-6

RNA analysis of EVs isolated by exoEasy kit and SEC qEV columns.  a  RNA concentration.  b  Representative bioanalyzer profiles of RNA isolated from the same donor and analyzed by RNA 6000 Pico Kit; the y-axis shows fluorescence units (FU) and the x-axis the nucleotide length (nt) of the RNA. Peaks at 25 nt is an internal standard.  c  Ratio of total amount of RNA (µg) to total amount of protein (µg). (mean ± SEM, n = 6; *p 
Figure Legend Snippet: RNA analysis of EVs isolated by exoEasy kit and SEC qEV columns. a RNA concentration. b Representative bioanalyzer profiles of RNA isolated from the same donor and analyzed by RNA 6000 Pico Kit; the y-axis shows fluorescence units (FU) and the x-axis the nucleotide length (nt) of the RNA. Peaks at 25 nt is an internal standard. c Ratio of total amount of RNA (µg) to total amount of protein (µg). (mean ± SEM, n = 6; *p 

Techniques Used: Isolation, Size-exclusion Chromatography, Concentration Assay, Fluorescence

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

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

Journal: Oncotarget

doi: 10.18632/oncotarget.6540

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

Techniques Used: Fluorescence

6) Product Images from "Gene expression analysis on small numbers of invasive cells collected by chemotaxis from primary mammary tumors of the mouse"

Article Title: Gene expression analysis on small numbers of invasive cells collected by chemotaxis from primary mammary tumors of the mouse

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-3-13

The RNA from 200 and 400 cells is of equal amount and quality of RNA purified by conventional methods.  Figure   5A : Total RNA isolated from microneedle collected cells was checked using Agilent Bioanalyzer and RNA 6000 Pico kit. The size distribution and rRNA ratio (28S/18S = 2.7) indicates good intactness of RNA sample. Figure   5B : A standard curve was generated for the CT value of a known quantity of RNA from a specific cell number using the β-actin primers for real-time PCR. The RNA from 200 and 400 cells were amplified by the SMART PCR method and run identically by real-time PCR. The CT values for the amplified RNA fall on the curve showing that the appropriate amount of the house keeping gene is present in the amplified sample. The CT values for the amplified samples are designated by the red asterisks.
Figure Legend Snippet: The RNA from 200 and 400 cells is of equal amount and quality of RNA purified by conventional methods. Figure 5A : Total RNA isolated from microneedle collected cells was checked using Agilent Bioanalyzer and RNA 6000 Pico kit. The size distribution and rRNA ratio (28S/18S = 2.7) indicates good intactness of RNA sample. Figure 5B : A standard curve was generated for the CT value of a known quantity of RNA from a specific cell number using the β-actin primers for real-time PCR. The RNA from 200 and 400 cells were amplified by the SMART PCR method and run identically by real-time PCR. The CT values for the amplified RNA fall on the curve showing that the appropriate amount of the house keeping gene is present in the amplified sample. The CT values for the amplified samples are designated by the red asterisks.

Techniques Used: Purification, Isolation, Generated, Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

7) Product Images from "Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy Donors"

Article Title: Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy Donors

Journal: Disease Markers

doi: 10.1155/2019/6852917

Quality of small RNA isolated from exosomes. Representative electropherogram of RNA quality extracted from exosomes derived of (a) healthy donors and (b) BC patients. All samples were analyzed by the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with the RNA 6000 Pico Kit (Agilent Technologies). The Y -axis represents fluorescence units (FU) and the X -axis shows nucleotides (nt). The peak observed between 25 nt and 200 nt correspond to the small RNA region. Peaks in the regions around 1500 nt and 4000 nt relative to 18S and 28S ribosomal RNAs, respectively, were not or barely detected. The peak of 25 nt refers to the marker. (c) Virtual gel of the data is shown in the electropherograms.
Figure Legend Snippet: Quality of small RNA isolated from exosomes. Representative electropherogram of RNA quality extracted from exosomes derived of (a) healthy donors and (b) BC patients. All samples were analyzed by the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with the RNA 6000 Pico Kit (Agilent Technologies). The Y -axis represents fluorescence units (FU) and the X -axis shows nucleotides (nt). The peak observed between 25 nt and 200 nt correspond to the small RNA region. Peaks in the regions around 1500 nt and 4000 nt relative to 18S and 28S ribosomal RNAs, respectively, were not or barely detected. The peak of 25 nt refers to the marker. (c) Virtual gel of the data is shown in the electropherograms.

Techniques Used: Isolation, Derivative Assay, Fluorescence, Marker

8) Product Images from "Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum"

Article Title: Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2017.3080

Bioanalyzer analysis of total exosomal RNA (ExoRNA) by Agilent RNA Pico chip. The experiment was repeated three times and the trend was the same, thus one of the results is shown. The RNA 6000 ladder standard (in the first lane) contains six RNA fragments ranging in size from 0.2 to 6 kb. Representative bands of cellular RNA (in the second lane) showed 5S (120 nt), 18S (1,900 nt) and 28S rRNA (4,700 nt). In bands of exoRNA from cell culture medium (CCM), almost all of the samples showed an obvious band in the small RNA area. Among the five combination methods, Route_4 and Route_5 (labeled by **) showed a narrow size distribution pattern of small RNA around 100 nt. Some longer RNA species, including 18S and 28S ribosomal RNA, were found in bands obtained using Route_1, Route_2 and Route_3 (labeled by *). For exoRNA from serum, Route_e (labeled by **) had the most obvious band in the position of small RNA, and was followed by Route_b, Route_c and Route_d (labeled by *). No visible bands could be found in samples from Route_a and Route_f.
Figure Legend Snippet: Bioanalyzer analysis of total exosomal RNA (ExoRNA) by Agilent RNA Pico chip. The experiment was repeated three times and the trend was the same, thus one of the results is shown. The RNA 6000 ladder standard (in the first lane) contains six RNA fragments ranging in size from 0.2 to 6 kb. Representative bands of cellular RNA (in the second lane) showed 5S (120 nt), 18S (1,900 nt) and 28S rRNA (4,700 nt). In bands of exoRNA from cell culture medium (CCM), almost all of the samples showed an obvious band in the small RNA area. Among the five combination methods, Route_4 and Route_5 (labeled by **) showed a narrow size distribution pattern of small RNA around 100 nt. Some longer RNA species, including 18S and 28S ribosomal RNA, were found in bands obtained using Route_1, Route_2 and Route_3 (labeled by *). For exoRNA from serum, Route_e (labeled by **) had the most obvious band in the position of small RNA, and was followed by Route_b, Route_c and Route_d (labeled by *). No visible bands could be found in samples from Route_a and Route_f.

Techniques Used: Chromatin Immunoprecipitation, Cell Culture, Labeling

9) Product Images from "A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis"

Article Title: A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis

Journal:

doi: 10.1128/IAI.00417-16

Contribution of the U1406A mutation to ribosome maturation. (A) Ratio of 17S to 16S rRNA in NCGM2242 and Erdman cells. The leader (5′ end) and trailer (3′ end) sequences of 17S rRNA are digested during the ribosome maturation process; 16S rRNA sequences were measured via qRT-PCR. Error bars indicate standard deviations. Differences were statistically significant (**, P < 0.01) based on a one-way ANOVA. (B) Ribosome populations in NCGM2242 and Erdman cells. The horizontal axis was adjusted by the migration distance of an RNA size marker and the internal control in the Agilent RNA 6000 Pico kit. nt, nucleotides.
Figure Legend Snippet: Contribution of the U1406A mutation to ribosome maturation. (A) Ratio of 17S to 16S rRNA in NCGM2242 and Erdman cells. The leader (5′ end) and trailer (3′ end) sequences of 17S rRNA are digested during the ribosome maturation process; 16S rRNA sequences were measured via qRT-PCR. Error bars indicate standard deviations. Differences were statistically significant (**, P < 0.01) based on a one-way ANOVA. (B) Ribosome populations in NCGM2242 and Erdman cells. The horizontal axis was adjusted by the migration distance of an RNA size marker and the internal control in the Agilent RNA 6000 Pico kit. nt, nucleotides.

Techniques Used: Mutagenesis, Quantitative RT-PCR, Migration, Marker

10) Product Images from "Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes"

Article Title: Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes

Journal: BMC Genomics

doi: 10.1186/s12864-015-1225-x

Experimental infection, parasite isolation, and RNA preparation. A)  Immunofluorescence assay using sheep immune serum against  T. gondii  revealed numerous infected enterocytes. Nuclei are counterstained with DAPI. Shown in the top panel is a 20x magnification of a section of the small intestine (bar = 100 μM) where villi are visible. The bottom panel at 100x magnification shows a schizont containing several merozoites (scale bar = 5 μm).  B)  Enterocytes containing CZ-strain merozoite stages were stripped away selectively, leaving the villus structure and the cells of the  lamina propria  intact. Histology section (Hematoxilin    Eosin stained) showing stripped villi at day 5 post infection.  C)  Microscopic examination of parasites in the detergent washed preparation showed only merozoite stages.  D)  Quality control of total RNA extracted from parasite preparations separated on an Agilent RNA 6000 Pico Chip. The bands generated by host 28S/18S ribosomal RNA (arrowheads) and parasite 26S/18S ribosomal RNA (arrows) as well as a size marker are indicated. The samples analyzed were: raw, unprocessed material from scraped intestinal lining; Tween 80, material that was syringe-passaged and washed twice with PBS/0.05% Tween 80; Percoll, highly enriched parasite fraction after detergent treatment and Percoll gradient centrifugation; Tachy, RNA prepared from tachyzoites grown in cell culture with human foreskin fibroblasts as host cells.
Figure Legend Snippet: Experimental infection, parasite isolation, and RNA preparation. A) Immunofluorescence assay using sheep immune serum against T. gondii revealed numerous infected enterocytes. Nuclei are counterstained with DAPI. Shown in the top panel is a 20x magnification of a section of the small intestine (bar = 100 μM) where villi are visible. The bottom panel at 100x magnification shows a schizont containing several merozoites (scale bar = 5 μm). B) Enterocytes containing CZ-strain merozoite stages were stripped away selectively, leaving the villus structure and the cells of the lamina propria intact. Histology section (Hematoxilin Eosin stained) showing stripped villi at day 5 post infection. C) Microscopic examination of parasites in the detergent washed preparation showed only merozoite stages. D) Quality control of total RNA extracted from parasite preparations separated on an Agilent RNA 6000 Pico Chip. The bands generated by host 28S/18S ribosomal RNA (arrowheads) and parasite 26S/18S ribosomal RNA (arrows) as well as a size marker are indicated. The samples analyzed were: raw, unprocessed material from scraped intestinal lining; Tween 80, material that was syringe-passaged and washed twice with PBS/0.05% Tween 80; Percoll, highly enriched parasite fraction after detergent treatment and Percoll gradient centrifugation; Tachy, RNA prepared from tachyzoites grown in cell culture with human foreskin fibroblasts as host cells.

Techniques Used: Infection, Isolation, Immunofluorescence, Staining, Chromatin Immunoprecipitation, Generated, Marker, Gradient Centrifugation, Cell Culture

11) Product Images from "A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies"

Article Title: A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies

Journal: Scientific Reports

doi: 10.1038/s41598-017-14264-5

Bioanalyzer electropherogram analysis of fragmentation time-points of leukocyte RNA. Arbitrary fluorescence units (FU) are plotted as a function of RNA size in nucleotides (nt). ( A ) Analysis of fragmentation time-points 15–180 min with Agilent RNA 6000 Pico Kit shows the expected ribosomal RNA peaks in non-fragmented sample (in red), whereas after all fragmentation time-points the majority of RNAs are below 200 nt in size. ( B ) Analysis of fragmentation time-points from 45 to 420 min with Agilent Small RNA Kit shows that the majority of RNAs are smaller than 40 nt in size for all time-points, and that a plateau is reached after approximately 180 min, as no further reduction in size is observed with longer fragmentation times. For the longer fragmentation periods, reduced amounts of RNA (marked with a star) were used to better simulate the enzymatic kinetics in the presence of less (but still detectable) RNA, and the plateau region is still the same.
Figure Legend Snippet: Bioanalyzer electropherogram analysis of fragmentation time-points of leukocyte RNA. Arbitrary fluorescence units (FU) are plotted as a function of RNA size in nucleotides (nt). ( A ) Analysis of fragmentation time-points 15–180 min with Agilent RNA 6000 Pico Kit shows the expected ribosomal RNA peaks in non-fragmented sample (in red), whereas after all fragmentation time-points the majority of RNAs are below 200 nt in size. ( B ) Analysis of fragmentation time-points from 45 to 420 min with Agilent Small RNA Kit shows that the majority of RNAs are smaller than 40 nt in size for all time-points, and that a plateau is reached after approximately 180 min, as no further reduction in size is observed with longer fragmentation times. For the longer fragmentation periods, reduced amounts of RNA (marked with a star) were used to better simulate the enzymatic kinetics in the presence of less (but still detectable) RNA, and the plateau region is still the same.

Techniques Used: Fluorescence

12) Product Images from "Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography"

Article Title: Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography

Journal: Journal of Extracellular Vesicles

doi: 10.1080/20013078.2017.1294340

Total RNA profiles for human and bovine MEV and cream fractions. Total RNA contained in fluff (red) and serum (green) MEV fractions were isolated from SEC F1/S1 peaks concentrated by reverse osmosis. Cream was sampled from the first centrifugation of raw milk. RNA extracts were analysed on Agilent RNA 6000 Pico chips using an Agilent Bioanalyzer. The total MEV RNA content was related to the initial start volume of milk (ng ml –1  milk). The peak at 25 nt is an internal standard. Abbreviations: FU, fluorescence units; nt, nucleotides; hF1, human fluff MEV; hS1, human milk serum MEV; bF1, bovine fluff MEV; bS1, bovine milk serum MEV.
Figure Legend Snippet: Total RNA profiles for human and bovine MEV and cream fractions. Total RNA contained in fluff (red) and serum (green) MEV fractions were isolated from SEC F1/S1 peaks concentrated by reverse osmosis. Cream was sampled from the first centrifugation of raw milk. RNA extracts were analysed on Agilent RNA 6000 Pico chips using an Agilent Bioanalyzer. The total MEV RNA content was related to the initial start volume of milk (ng ml –1 milk). The peak at 25 nt is an internal standard. Abbreviations: FU, fluorescence units; nt, nucleotides; hF1, human fluff MEV; hS1, human milk serum MEV; bF1, bovine fluff MEV; bS1, bovine milk serum MEV.

Techniques Used: Isolation, Size-exclusion Chromatography, Centrifugation, Fluorescence

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

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

Journal: BMC Research Notes

doi: 10.1186/1756-0500-7-62

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

Techniques Used: Concentration Assay, Produced

14) Product Images from "Cells release subpopulations of exosomes with distinct molecular and biological properties"

Article Title: Cells release subpopulations of exosomes with distinct molecular and biological properties

Journal: Scientific Reports

doi: 10.1038/srep22519

EV subpopulations have different RNA profiles. RNA from MV, LD-Exo and HD-Exo was extracted using Trizol and analyzed using capillary electrophoresis with the Agilent RNA 6000 Pico chip (left panel) and Agilent small RNA chip (right panel) on an Agilent 2100 Bioanalyzer®. The y-axis of the electropherograms represents fluorescence units (FU) and the x-axis represents the nucleotide length of the RNA (nt). Peaks at 25 nt (left panels) or at 4 nt (right panels) represent internal standards. Data shown are representative of two independent experiments.
Figure Legend Snippet: EV subpopulations have different RNA profiles. RNA from MV, LD-Exo and HD-Exo was extracted using Trizol and analyzed using capillary electrophoresis with the Agilent RNA 6000 Pico chip (left panel) and Agilent small RNA chip (right panel) on an Agilent 2100 Bioanalyzer®. The y-axis of the electropherograms represents fluorescence units (FU) and the x-axis represents the nucleotide length of the RNA (nt). Peaks at 25 nt (left panels) or at 4 nt (right panels) represent internal standards. Data shown are representative of two independent experiments.

Techniques Used: Electrophoresis, Chromatin Immunoprecipitation, Fluorescence

Related Articles

Centrifugation:

Article Title: Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes
Article Snippet: Cell suspensions were then passed through a QIAshredder (QIAGEN) column by centrifugation at ≥ 8000 g for 1 min. .. The quality of the RNA was analysed using the Agilent RNA 6000 Pico Kit (Agilent) and a Bioanalyzer 2100 (Agilent) (Figure D).

Article Title: Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography
Article Snippet: Cream (2 mg) sampled from the first centrifugation of raw milk fractions was additionally added Lysis Additive (Exiqon) to aid the RNA isolation. .. RNA isolates were analysed in triplicate by capillary electrophoresis using the Agilent RNA 6000 Pico kit on an Agilent 2100 Bioanlyzer® (Agilent Technologies) as previously described.

Article Title: Development of Genetic System to Inactivate a Borrelia turicatae Surface Protein Selectively Produced within the Salivary Glands of the Arthropod Vector
Article Snippet: B. turicatae was pelleted by centrifugation at 15,000× g for 20 min, supernatant was removed, and the spirochete-enriched pellet was suspended in 100 µl of RNAlater RNA Stabilizing Reagent (Qiagen), flash frozen using liquid nitrogen, and stored at −80°C until RNA was extracted. .. RNA integrity was determined using the Agilent RNA 6000 Pico kit and the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbroon, Germany) following the manufacturer's instructions.

Amplification:

Article Title: Loss of BAP1 function leads to EZH2-dependent transformation
Article Snippet: Total RNA from 200–500K cells was extracted using TRIzol RNA Isolation Reagents (cat# 15596-026, Life Technologies). .. Quality of RNA was ensured before amplification by analyzing 20–50 pg of each sample using the RNA 6000 pico kit and a bioAnalyzer (Agilent). .. 10 ng of high quality (RIN > 8) total RNA was subsequently amplified using the SMARTer® Universal Low Input RNA Kit for Sequencing (Clonetech Laboratory, cat# 634940) according to instructions provided by the manufacturer.

Cytometry:

Article Title: LncRNA profiling of human lymphoid progenitors reveals transcriptional divergence of B and T lineages
Article Snippet: Cells cryopreserved post MACS were thawed and used for isolation of subsets by flow cytometry for the other replicate of the thymus. .. The RNA 6000 Pico kit (Agilent technologies) was used to assess RNA integrity prior to library preparation.

Construct:

Article Title: Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer
Article Snippet: Index files were constructed using the bismark_genome_preparation command and the entire reference genome. .. RNA quality was controlled using the Agilent RNA 6000 Pico Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California) according to the manufacturer’s recommendations.

Electrophoresis:

Article Title: Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography
Article Snippet: All isolation procedures were performed according to the manufacturer’s protocol. .. RNA isolates were analysed in triplicate by capillary electrophoresis using the Agilent RNA 6000 Pico kit on an Agilent 2100 Bioanlyzer® (Agilent Technologies) as previously described. .. [ ] RNA concentration estimates were based on the concentrations calculated by the Agilent software, and the volume of milk from which the MEV fractions were isolated.

Article Title: Oxygen control of breathing by an olfactory receptor activated by lactate
Article Snippet: Tissue pieces were disrupted in a guanidine-isothiocyanate lysis buffer (Buffer RLT, Qiagen) using a glass tissue grinder (Corning), homogenized using a 20G needle and syringe, and purified by silica-membrane columns. .. RNA quality was assessed by electrophoresis on a Bioanalyzer using the RNA 6000 Pico Kit (Agilent). .. The average RNA Integrity Numbers (RIN) for carotid body and adrenal medulla samples were 7.2 and 9.0, respectively.

Article Title: Cells release subpopulations of exosomes with distinct molecular and biological properties
Article Snippet: Isolated RNA was resuspended in RNase free water and RNA yield was determined with Quant-iT™ RiboGreen® RNA assay (Life Technologies) according to the manufacturer’s protocol. .. Quality and size of RNA in exosomes were determined using capillary electrophoresis with the Agilent RNA 6000 Pico kit and Agilent RNA small RNA kit on an Agilent 2100 Bioanalyzer® (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. .. H5V cells were seeded in 24 well plates at a density of 60,000 cells per well in DMEM (Life Technologies) supplemented with 10% HI FCS depleted of serum EVs.

Microarray:

Article Title: Haematopoietic stem cells require a highly regulated protein synthesis rate
Article Snippet: RNA from each sample was extracted with the RNeasy micro plus kit (Qiagen) into 14 μl of water. .. 2 μl of each sample was analysed in duplicate for 18S rRNA, 28S rRNA, and total RNA concentration using an Agilent RNA 6000 Pico kit and a Bioanalyzer (Agilent) at the UT Southwestern Genomics & Microarray Core. .. 50,000 cells of each population were sorted into PBS.

Incubation:

Article Title: A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis
Article Snippet: The lysates were filtered through 0.22-mm filters, 4 μl of DNase (Qiagen Japan) was added, and the samples were incubated for 10 min on ice. .. Ribosome fractions were analyzed by using the Agilent RNA 6000 Pico kit and Bioanalyzer 2100 apparatus (Agilent Technologies Japan, Tokyo, Japan) according to the manufacturer's protocols.

Article Title: Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy Donors
Article Snippet: After 3 minutes of incubation at room temperature, the protein precipitation solution BF was added. .. The quality of the RNA isolated was determined by an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with the RNA 6000 Pico Kit (Agilent Technologies) according to the manufacturer's protocol.

Formalin-fixed Paraffin-Embedded:

Article Title: Single-Cell Sequencing of iPSC-Dopamine Neurons Reconstructs Disease Progression and Identifies HDAC4 as a Regulator of Parkinson Cell Phenotypes
Article Snippet: RNA from bulk collected FACS sorted dopamine neurons was extracted using an FFPE RNA extraction kit (QIAGEN) as per manufacturer’s instructions, with minor modifications. .. RNA integrity analysis was analyzed using a 2100 bioanalyzer system and a RNA 6000 pico kit (Agilent).

Expressing:

Article Title: Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer
Article Snippet: RNA quality was controlled using the Agilent RNA 6000 Pico Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California) according to the manufacturer’s recommendations. .. RNA quality was controlled using the Agilent RNA 6000 Pico Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California) according to the manufacturer’s recommendations.

Genome Wide:

Article Title: Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes
Article Snippet: The quality of the RNA was analysed using the Agilent RNA 6000 Pico Kit (Agilent) and a Bioanalyzer 2100 (Agilent) (Figure D). .. RNA concentration was determined using a Qubit fluorometer (Invitrogen) together with the RNA assay (Invitrogen).

Western Blot:

Article Title: Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography
Article Snippet: RNA isolates were analysed in triplicate by capillary electrophoresis using the Agilent RNA 6000 Pico kit on an Agilent 2100 Bioanlyzer® (Agilent Technologies) as previously described. .. [ ] RNA concentration estimates were based on the concentrations calculated by the Agilent software, and the volume of milk from which the MEV fractions were isolated.

Flow Cytometry:

Article Title: LncRNA profiling of human lymphoid progenitors reveals transcriptional divergence of B and T lineages
Article Snippet: Cells cryopreserved post MACS were thawed and used for isolation of subsets by flow cytometry for the other replicate of the thymus. .. The RNA 6000 Pico kit (Agilent technologies) was used to assess RNA integrity prior to library preparation.

Infection:

Article Title: Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes
Article Snippet: Infected tissue and parasite samples were resuspended in 600 μl buffer RLT (QIAGEN) containing 10 μl/ml β-mercaptoethanol. .. The quality of the RNA was analysed using the Agilent RNA 6000 Pico Kit (Agilent) and a Bioanalyzer 2100 (Agilent) (Figure D).

Article Title: Development of Genetic System to Inactivate a Borrelia turicatae Surface Protein Selectively Produced within the Salivary Glands of the Arthropod Vector
Article Snippet: Paragraph title: RNA isolation and analysis of B. turicatae in infected mouse blood, ticks, and spirochetes in vitro cultivated at 22°C and 35°C ... RNA integrity was determined using the Agilent RNA 6000 Pico kit and the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbroon, Germany) following the manufacturer's instructions.

other:

Article Title: Ensuring good quality rna for quantitative real-time pcr isolated from renal proximal tubular cells using laser capture microdissection
Article Snippet: RIN was measured using Agilent 2100 Bioanalyzer with Agilent RNA 6000 Pico Kit.

Sequencing:

Article Title: A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies
Article Snippet: First, we used RNA fragmentation to increase the number of molecules as a means of reducing the formation of adaptor-dimer artifacts, which consume sequencing space and it is a known major issue when limiting amounts of nucleic acids are available for NGS library construction . .. As EV-derived RNA obtained from plasma cannot be generally visualized in the Bioanalyzer due to its very low amounts, the fragmentation time was optimized with RNA extracted from peripheral blood leukocytes, analyzed with the Agilent RNA 6000 Pico Kit.

Article Title: Loss of BAP1 function leads to EZH2-dependent transformation
Article Snippet: Paragraph title: RNA isolation, SMARTer amplification, Proton Transcriptome sequencing and analysis ... Quality of RNA was ensured before amplification by analyzing 20–50 pg of each sample using the RNA 6000 pico kit and a bioAnalyzer (Agilent).

Article Title: Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer
Article Snippet: Paragraph title: High-throughput sequencing ... RNA quality was controlled using the Agilent RNA 6000 Pico Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California) according to the manufacturer’s recommendations.

Magnetic Cell Separation:

Article Title: LncRNA profiling of human lymphoid progenitors reveals transcriptional divergence of B and T lineages
Article Snippet: Cells cryopreserved post MACS were thawed and used for isolation of subsets by flow cytometry for the other replicate of the thymus. .. The RNA 6000 Pico kit (Agilent technologies) was used to assess RNA integrity prior to library preparation.

RNA Sequencing Assay:

Article Title: Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes
Article Snippet: The quality of the RNA was analysed using the Agilent RNA 6000 Pico Kit (Agilent) and a Bioanalyzer 2100 (Agilent) (Figure D). .. RNA concentration was determined using a Qubit fluorometer (Invitrogen) together with the RNA assay (Invitrogen).

Article Title: LncRNA profiling of human lymphoid progenitors reveals transcriptional divergence of B and T lineages
Article Snippet: The RNA 6000 Pico kit (Agilent technologies) was used to assess RNA integrity prior to library preparation. .. The RNA 6000 Pico kit (Agilent technologies) was used to assess RNA integrity prior to library preparation.

Article Title: Loss of BAP1 function leads to EZH2-dependent transformation
Article Snippet: Quality of RNA was ensured before amplification by analyzing 20–50 pg of each sample using the RNA 6000 pico kit and a bioAnalyzer (Agilent). .. 10 ng of high quality (RIN > 8) total RNA was subsequently amplified using the SMARTer® Universal Low Input RNA Kit for Sequencing (Clonetech Laboratory, cat# 634940) according to instructions provided by the manufacturer.

Article Title: Single-Cell Sequencing of iPSC-Dopamine Neurons Reconstructs Disease Progression and Identifies HDAC4 as a Regulator of Parkinson Cell Phenotypes
Article Snippet: Paragraph title: RNA preparation of bulk RNA-seq samples ... RNA integrity analysis was analyzed using a 2100 bioanalyzer system and a RNA 6000 pico kit (Agilent).

Article Title: Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer
Article Snippet: The methylation level of each sampled cytosine was estimated as the number of reads reporting a C, divided by the total number of reads reporting a C or T. Transcriptome analysis by RNA-seq: Total RNA from dissected Alb-R26 Met tumours (n = 4) and control livers (n = 4) was processed for transcriptome analysis. .. RNA quality was controlled using the Agilent RNA 6000 Pico Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California) according to the manufacturer’s recommendations.

RNA HS Assay:

Article Title: Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes
Article Snippet: Where indicated, RNA or DNA was quantified using Qubit RNA HS Assay Kit and Qubit dsDNA HS Assay Kit (Life Technologies), respectively, on the Qubit 2.0 Fluorometer (Life Technologies). .. In addition, where indicated, fragment size distribution of RNA or DNA samples was assessed on the Agilent 2100 BioAnalyzer using the Agilent RNA 6000 Pico Kit or Agilent High Sensitivity DNA Kit (Agilent Technologies), respectively.

Methylation:

Article Title: Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer
Article Snippet: The methylation level of each sampled cytosine was estimated as the number of reads reporting a C, divided by the total number of reads reporting a C or T. Transcriptome analysis by RNA-seq: Total RNA from dissected Alb-R26 Met tumours (n = 4) and control livers (n = 4) was processed for transcriptome analysis. .. RNA quality was controlled using the Agilent RNA 6000 Pico Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California) according to the manufacturer’s recommendations.

Isolation:

Article Title: Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes
Article Snippet: Paragraph title: RNA isolation and quality control ... The quality of the RNA was analysed using the Agilent RNA 6000 Pico Kit (Agilent) and a Bioanalyzer 2100 (Agilent) (Figure D).

Article Title: Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography
Article Snippet: Paragraph title: RNA isolation and detection ... RNA isolates were analysed in triplicate by capillary electrophoresis using the Agilent RNA 6000 Pico kit on an Agilent 2100 Bioanlyzer® (Agilent Technologies) as previously described.

Article Title: Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma
Article Snippet: Paragraph title: RNA isolation from EVs ... RNA yield and size range were analyzed on an Agilent 2100 Bioanalyzer using the RNA 6000 Pico Kit and the Small RNA Kit (Agilent Technologies).

Article Title: Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum
Article Snippet: Paragraph title: ExoRNA isolation and RNA analyses ... The RNA yield and size distribution were analyzed using an Agilent 2100 Bioanalyzer with an RNA 6000 Pico kit (Agilent Technologies, Foster City, CA, USA).

Article Title: Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy Donors
Article Snippet: Next, isopropanol was added, and samples were loaded onto a column followed by 3 washes and the RNA elution in 50 μ l of RNase-free water. .. The quality of the RNA isolated was determined by an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with the RNA 6000 Pico Kit (Agilent Technologies) according to the manufacturer's protocol. .. For the analysis of the expression of miR-145, miR-155, and miR-382 in exosomes isolated from BC patients and healthy donors, 150 ng of total RNA was reverse-transcribed to cDNA using the qScript microRNA cDNA Synthesis Kit (Quanta BioSciences, Gaithersburg, MD).

Article Title: Cells release subpopulations of exosomes with distinct molecular and biological properties
Article Snippet: Isolated RNA was resuspended in RNase free water and RNA yield was determined with Quant-iT™ RiboGreen® RNA assay (Life Technologies) according to the manufacturer’s protocol. .. Quality and size of RNA in exosomes were determined using capillary electrophoresis with the Agilent RNA 6000 Pico kit and Agilent RNA small RNA kit on an Agilent 2100 Bioanalyzer® (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol.

Article Title: Exosomes from bulk and stem cells from human prostate cancer have a differential microRNA content that contributes cooperatively over local and pre-metastatic niche
Article Snippet: First, exosomes were lysed and after organic extraction, total RNA was isolated over glass-fiber filter and eluted. .. The quality of the total RNA was assessed by Agilent 2100 Bioanalyzer using an RNA 6000 Pico Kit (Agilent Technologies).

Article Title: LncRNA profiling of human lymphoid progenitors reveals transcriptional divergence of B and T lineages
Article Snippet: Paragraph title: Isolation and RNA extraction from bone marrow and thymic progenitors ... The RNA 6000 Pico kit (Agilent technologies) was used to assess RNA integrity prior to library preparation.

Article Title: Loss of BAP1 function leads to EZH2-dependent transformation
Article Snippet: Paragraph title: RNA isolation, SMARTer amplification, Proton Transcriptome sequencing and analysis ... Quality of RNA was ensured before amplification by analyzing 20–50 pg of each sample using the RNA 6000 pico kit and a bioAnalyzer (Agilent).

Article Title: Development of Genetic System to Inactivate a Borrelia turicatae Surface Protein Selectively Produced within the Salivary Glands of the Arthropod Vector
Article Snippet: Paragraph title: RNA isolation and analysis of B. turicatae in infected mouse blood, ticks, and spirochetes in vitro cultivated at 22°C and 35°C ... RNA integrity was determined using the Agilent RNA 6000 Pico kit and the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbroon, Germany) following the manufacturer's instructions.

Size-exclusion Chromatography:

Article Title: Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma
Article Snippet: Total RNA was isolated from concentrated pooled EV-rich SEC fractions or directly from exoEasy kit columns following the protocol of the exoRNeasy Serum/Plasma Kit (Qiagen). .. RNA yield and size range were analyzed on an Agilent 2100 Bioanalyzer using the RNA 6000 Pico Kit and the Small RNA Kit (Agilent Technologies).

Purification:

Article Title: Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes
Article Snippet: The RNA was then purified using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol (including an on-column DNA digest), and eluted in RNase-free water. .. The quality of the RNA was analysed using the Agilent RNA 6000 Pico Kit (Agilent) and a Bioanalyzer 2100 (Agilent) (Figure D).

Article Title: Oxygen control of breathing by an olfactory receptor activated by lactate
Article Snippet: Paragraph title: Carotid body and adrenal medulla RNA purification ... RNA quality was assessed by electrophoresis on a Bioanalyzer using the RNA 6000 Pico Kit (Agilent).

Polymerase Chain Reaction:

Article Title: Loss of BAP1 function leads to EZH2-dependent transformation
Article Snippet: Quality of RNA was ensured before amplification by analyzing 20–50 pg of each sample using the RNA 6000 pico kit and a bioAnalyzer (Agilent). .. 10 ng of high quality (RIN > 8) total RNA was subsequently amplified using the SMARTer® Universal Low Input RNA Kit for Sequencing (Clonetech Laboratory, cat# 634940) according to instructions provided by the manufacturer.

Lysis:

Article Title: Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography
Article Snippet: Cream (2 mg) sampled from the first centrifugation of raw milk fractions was additionally added Lysis Additive (Exiqon) to aid the RNA isolation. .. RNA isolates were analysed in triplicate by capillary electrophoresis using the Agilent RNA 6000 Pico kit on an Agilent 2100 Bioanlyzer® (Agilent Technologies) as previously described.

Article Title: Detection of circulating miRNAs: comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients
Article Snippet: Briefly, 5 volumes of QIAzol Lysis Reagent were added to each sample. .. The quantity and quality of RNA was assessed using Agilent 2100 Bioanalyzer and RNA 6000 Pico Kit (Agilent technologies, # 5067–1513).

Article Title: Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy Donors
Article Snippet: Briefly, exosomes were resuspended in 200 μ l of RNase-free water, then 60 μ l of lysis solution BF was added. .. The quality of the RNA isolated was determined by an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with the RNA 6000 Pico Kit (Agilent Technologies) according to the manufacturer's protocol.

Article Title: Oxygen control of breathing by an olfactory receptor activated by lactate
Article Snippet: Tissue pieces were disrupted in a guanidine-isothiocyanate lysis buffer (Buffer RLT, Qiagen) using a glass tissue grinder (Corning), homogenized using a 20G needle and syringe, and purified by silica-membrane columns. .. RNA quality was assessed by electrophoresis on a Bioanalyzer using the RNA 6000 Pico Kit (Agilent).

Real-time Polymerase Chain Reaction:

Article Title: LncRNA profiling of human lymphoid progenitors reveals transcriptional divergence of B and T lineages
Article Snippet: The RNA 6000 Pico kit (Agilent technologies) was used to assess RNA integrity prior to library preparation. .. The RNA 6000 Pico kit (Agilent technologies) was used to assess RNA integrity prior to library preparation.

RNA Extraction:

Article Title: Detection of circulating miRNAs: comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients
Article Snippet: Paragraph title: RNA extraction ... The quantity and quality of RNA was assessed using Agilent 2100 Bioanalyzer and RNA 6000 Pico Kit (Agilent technologies, # 5067–1513).

Article Title: Exosomal miRNAs from Peritoneum Lavage Fluid as Potential Prognostic Biomarkers of Peritoneal Metastasis in Gastric Cancer
Article Snippet: Paragraph title: Total RNA extraction and analysis ... Prior to the analysis, total RNA was prepared using an Agilent RNA 6000 Pico kit (Agilent Technologies) according to the manufacturer's protocol.

Article Title: Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy Donors
Article Snippet: The miRCURY™ RNA isolation kit for biofluids (Exiqon, Vedbaek, Denmark) was used for RNA extraction. .. The quality of the RNA isolated was determined by an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) with the RNA 6000 Pico Kit (Agilent Technologies) according to the manufacturer's protocol.

Article Title: Cells release subpopulations of exosomes with distinct molecular and biological properties
Article Snippet: Paragraph title: RNA extraction and profiling ... Quality and size of RNA in exosomes were determined using capillary electrophoresis with the Agilent RNA 6000 Pico kit and Agilent RNA small RNA kit on an Agilent 2100 Bioanalyzer® (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol.

Article Title: LncRNA profiling of human lymphoid progenitors reveals transcriptional divergence of B and T lineages
Article Snippet: Paragraph title: Isolation and RNA extraction from bone marrow and thymic progenitors ... The RNA 6000 Pico kit (Agilent technologies) was used to assess RNA integrity prior to library preparation.

Article Title: Single-Cell Sequencing of iPSC-Dopamine Neurons Reconstructs Disease Progression and Identifies HDAC4 as a Regulator of Parkinson Cell Phenotypes
Article Snippet: RNA from bulk collected FACS sorted dopamine neurons was extracted using an FFPE RNA extraction kit (QIAGEN) as per manufacturer’s instructions, with minor modifications. .. RNA integrity analysis was analyzed using a 2100 bioanalyzer system and a RNA 6000 pico kit (Agilent).

In Vitro:

Article Title: Development of Genetic System to Inactivate a Borrelia turicatae Surface Protein Selectively Produced within the Salivary Glands of the Arthropod Vector
Article Snippet: Paragraph title: RNA isolation and analysis of B. turicatae in infected mouse blood, ticks, and spirochetes in vitro cultivated at 22°C and 35°C ... RNA integrity was determined using the Agilent RNA 6000 Pico kit and the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbroon, Germany) following the manufacturer's instructions.

Next-Generation Sequencing:

Article Title: A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies
Article Snippet: Paragraph title: Optimization of RNA input for NGS library-construction ... As EV-derived RNA obtained from plasma cannot be generally visualized in the Bioanalyzer due to its very low amounts, the fragmentation time was optimized with RNA extracted from peripheral blood leukocytes, analyzed with the Agilent RNA 6000 Pico Kit.

Spectrophotometry:

Article Title: Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum
Article Snippet: The RNA concentration was assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). .. The RNA yield and size distribution were analyzed using an Agilent 2100 Bioanalyzer with an RNA 6000 Pico kit (Agilent Technologies, Foster City, CA, USA).

Produced:

Article Title: Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes
Article Snippet: The quality of the RNA was analysed using the Agilent RNA 6000 Pico Kit (Agilent) and a Bioanalyzer 2100 (Agilent) (Figure D). .. RNA concentration was determined using a Qubit fluorometer (Invitrogen) together with the RNA assay (Invitrogen).

Concentration Assay:

Article Title: Exosomal miRNAs from Peritoneum Lavage Fluid as Potential Prognostic Biomarkers of Peritoneal Metastasis in Gastric Cancer
Article Snippet: The quality, concentration, and size of total exosomal RNA were assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Foster City, CA). .. Prior to the analysis, total RNA was prepared using an Agilent RNA 6000 Pico kit (Agilent Technologies) according to the manufacturer's protocol.

Article Title: Comparison of isolation methods of exosomes and exosomal RNA from cell culture medium and serum
Article Snippet: The RNA concentration was assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). .. The RNA yield and size distribution were analyzed using an Agilent 2100 Bioanalyzer with an RNA 6000 Pico kit (Agilent Technologies, Foster City, CA, USA).

Article Title: Exosomes from bulk and stem cells from human prostate cancer have a differential microRNA content that contributes cooperatively over local and pre-metastatic niche
Article Snippet: The quality of the total RNA was assessed by Agilent 2100 Bioanalyzer using an RNA 6000 Pico Kit (Agilent Technologies). .. The quality of the total RNA was assessed by Agilent 2100 Bioanalyzer using an RNA 6000 Pico Kit (Agilent Technologies).

Article Title: Haematopoietic stem cells require a highly regulated protein synthesis rate
Article Snippet: RNA from each sample was extracted with the RNeasy micro plus kit (Qiagen) into 14 μl of water. .. 2 μl of each sample was analysed in duplicate for 18S rRNA, 28S rRNA, and total RNA concentration using an Agilent RNA 6000 Pico kit and a Bioanalyzer (Agilent) at the UT Southwestern Genomics & Microarray Core. .. 50,000 cells of each population were sorted into PBS.

Thin Layer Chromatography:

Article Title: Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography
Article Snippet: RNA isolates were analysed in triplicate by capillary electrophoresis using the Agilent RNA 6000 Pico kit on an Agilent 2100 Bioanlyzer® (Agilent Technologies) as previously described. .. [ ] RNA concentration estimates were based on the concentrations calculated by the Agilent software, and the volume of milk from which the MEV fractions were isolated.

Liquid Chromatography with Mass Spectroscopy:

Article Title: Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography
Article Snippet: RNA isolates were analysed in triplicate by capillary electrophoresis using the Agilent RNA 6000 Pico kit on an Agilent 2100 Bioanlyzer® (Agilent Technologies) as previously described. .. [ ] RNA concentration estimates were based on the concentrations calculated by the Agilent software, and the volume of milk from which the MEV fractions were isolated.

High Throughput Screening Assay:

Article Title: Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer
Article Snippet: Paragraph title: High-throughput sequencing ... RNA quality was controlled using the Agilent RNA 6000 Pico Kit and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California) according to the manufacturer’s recommendations.

FACS:

Article Title: Loss of BAP1 function leads to EZH2-dependent transformation
Article Snippet: Bone marrow cells were FACS sorted for GMPs (Lin– c-Kit+ Sca1– CD34+ Fcγ+ ) using the FACS Aria. .. Quality of RNA was ensured before amplification by analyzing 20–50 pg of each sample using the RNA 6000 pico kit and a bioAnalyzer (Agilent).

Article Title: Single-Cell Sequencing of iPSC-Dopamine Neurons Reconstructs Disease Progression and Identifies HDAC4 as a Regulator of Parkinson Cell Phenotypes
Article Snippet: RNA from bulk collected FACS sorted dopamine neurons was extracted using an FFPE RNA extraction kit (QIAGEN) as per manufacturer’s instructions, with minor modifications. .. RNA integrity analysis was analyzed using a 2100 bioanalyzer system and a RNA 6000 pico kit (Agilent).

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    Agilent technologies rna 6000 pico kit
    Depletion of rRNA from RNA samples isolated from dual species  P. aeruginosa / S. aureus  cultures. A total of 2 μg of DNAse-treated RNA, isolated form a dual species  P. aeruginosa  PAO1 and  S. aureus  ATCC6538 culture, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( A ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( B ) MICROBExpress or ( C ) Ribo-Zero kits are shown. ( D ) Percent of rRNA contamination as reported by the Agilent Bioanalyzer 2100 Expert Software in RNA samples from single and dual species cultures of  P. aeruginosa  and/or  S. aureus  treated with the Ribo-Zero or MICROBExpress kits. Untreated input RNA (Total) or RNA processed via Ribo-Zero or MICROBExpress treatment was subjected to qPCR analysis of  P. aeruginosa  ( E , G ) or  S. aureus  ( F , H ) rRNA transcript abundance. ( E , F ) qPCR threshold cycle (Cq) for the indicated rRNA transcripts. ( G , H ) Copy numbers of  P. aeruginosa  or  S. aureus  16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.
    Rna 6000 Pico Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Depletion of rRNA from RNA samples isolated from dual species  P. aeruginosa / S. aureus  cultures. A total of 2 μg of DNAse-treated RNA, isolated form a dual species  P. aeruginosa  PAO1 and  S. aureus  ATCC6538 culture, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( A ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( B ) MICROBExpress or ( C ) Ribo-Zero kits are shown. ( D ) Percent of rRNA contamination as reported by the Agilent Bioanalyzer 2100 Expert Software in RNA samples from single and dual species cultures of  P. aeruginosa  and/or  S. aureus  treated with the Ribo-Zero or MICROBExpress kits. Untreated input RNA (Total) or RNA processed via Ribo-Zero or MICROBExpress treatment was subjected to qPCR analysis of  P. aeruginosa  ( E , G ) or  S. aureus  ( F , H ) rRNA transcript abundance. ( E , F ) qPCR threshold cycle (Cq) for the indicated rRNA transcripts. ( G , H ) Copy numbers of  P. aeruginosa  or  S. aureus  16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

    Journal: Scientific Reports

    Article Title: Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

    doi: 10.1038/srep41114

    Figure Lengend Snippet: Depletion of rRNA from RNA samples isolated from dual species P. aeruginosa / S. aureus cultures. A total of 2 μg of DNAse-treated RNA, isolated form a dual species P. aeruginosa PAO1 and S. aureus ATCC6538 culture, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( A ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( B ) MICROBExpress or ( C ) Ribo-Zero kits are shown. ( D ) Percent of rRNA contamination as reported by the Agilent Bioanalyzer 2100 Expert Software in RNA samples from single and dual species cultures of P. aeruginosa and/or S. aureus treated with the Ribo-Zero or MICROBExpress kits. Untreated input RNA (Total) or RNA processed via Ribo-Zero or MICROBExpress treatment was subjected to qPCR analysis of P. aeruginosa ( E , G ) or S. aureus ( F , H ) rRNA transcript abundance. ( E , F ) qPCR threshold cycle (Cq) for the indicated rRNA transcripts. ( G , H ) Copy numbers of P. aeruginosa or S. aureus 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

    Article Snippet: In addition, where indicated, fragment size distribution of RNA or DNA samples was assessed on the Agilent 2100 BioAnalyzer using the Agilent RNA 6000 Pico Kit or Agilent High Sensitivity DNA Kit (Agilent Technologies), respectively.

    Techniques: Isolation, Software, Real-time Polymerase Chain Reaction, Standard Deviation

    Quantitative and qualitative comparison of RNA recovered following rRNA depletion. A total of 4 μg of DNAse-treated RNA, isolated form 3-day-old  P. aeruginosa  PAO1 biofilms, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit and the Life Technologies RiboMinus Transcriptome Isolation Kit, Bacteria. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using Qubit fluorimetric quantitation with the Qubit RNA HS Assay Kit. Yields are reported as total RNA recovered ( A ) and as percentage of the input RNA ( B ). The RNA samples were also assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( C ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( D ) MICROBExpress, ( E ) RiboMinus, or ( F ) Ribo-Zero kits are shown. Dashed lines indicate peaks corresponding to 16S and 23S RNA traces, which were detected in total RNA, MICROBExpress, and RiboMins samples. ( G ) The area of 16S and 23S rRNA peaks as percent of the total detected RNA was estimated, as determined using the 2100 Expert Software. RFU, relative fluorescence units. Experiments were repeated using three biological replicates.

    Journal: Scientific Reports

    Article Title: Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

    doi: 10.1038/srep41114

    Figure Lengend Snippet: Quantitative and qualitative comparison of RNA recovered following rRNA depletion. A total of 4 μg of DNAse-treated RNA, isolated form 3-day-old P. aeruginosa PAO1 biofilms, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit and the Life Technologies RiboMinus Transcriptome Isolation Kit, Bacteria. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using Qubit fluorimetric quantitation with the Qubit RNA HS Assay Kit. Yields are reported as total RNA recovered ( A ) and as percentage of the input RNA ( B ). The RNA samples were also assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of ( C ) starting total RNA material and aliquots of the RNA samples that have been processed using the ( D ) MICROBExpress, ( E ) RiboMinus, or ( F ) Ribo-Zero kits are shown. Dashed lines indicate peaks corresponding to 16S and 23S RNA traces, which were detected in total RNA, MICROBExpress, and RiboMins samples. ( G ) The area of 16S and 23S rRNA peaks as percent of the total detected RNA was estimated, as determined using the 2100 Expert Software. RFU, relative fluorescence units. Experiments were repeated using three biological replicates.

    Article Snippet: In addition, where indicated, fragment size distribution of RNA or DNA samples was assessed on the Agilent 2100 BioAnalyzer using the Agilent RNA 6000 Pico Kit or Agilent High Sensitivity DNA Kit (Agilent Technologies), respectively.

    Techniques: Isolation, Quantitation Assay, RNA HS Assay, Software, Fluorescence

    rRNA depletion treatments of RNA derived from planktonic  P. aeruginosa  PAO1 and  Staphylococcus aureus  ATCC6538 cells. A total of 2 μg of DNAse-treated RNA, isolated from exponential phase  P. aeruginosa  PAO1 or  Staphylococcus aureus  ATCC6538 cells, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of starting total RNA material and aliquots of the RNA samples that have been processed using the MICROBExpress or Ribo-Zero kits are shown for the  P. aeruginosa  ( A ) and  S. aureus  ( B ) samples. The samples were subsequently subjected to qPCR analysis of rRNA transcript abundance, with qPCR threshold cycle (Cq) for the indicated rRNA transcripts shown for  P. aeruginosa  ( C ) and  S. aureus  ( E ). Copy numbers of  P. aeruginosa  ( D ) and  S. aureus  ( F ) 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

    Journal: Scientific Reports

    Article Title: Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes

    doi: 10.1038/srep41114

    Figure Lengend Snippet: rRNA depletion treatments of RNA derived from planktonic P. aeruginosa PAO1 and Staphylococcus aureus ATCC6538 cells. A total of 2 μg of DNAse-treated RNA, isolated from exponential phase P. aeruginosa PAO1 or Staphylococcus aureus ATCC6538 cells, was subjected to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria) or Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit. Following rRNA depletion and ethanol/acetate precipitation and resuspension in equal volumes of water, the RNA was assessed using the Bioanalyzer RNA 6000 Pico kit. Representative electropherograms of starting total RNA material and aliquots of the RNA samples that have been processed using the MICROBExpress or Ribo-Zero kits are shown for the P. aeruginosa ( A ) and S. aureus ( B ) samples. The samples were subsequently subjected to qPCR analysis of rRNA transcript abundance, with qPCR threshold cycle (Cq) for the indicated rRNA transcripts shown for P. aeruginosa ( C ) and S. aureus ( E ). Copy numbers of P. aeruginosa ( D ) and S. aureus ( F ) 16S, 23 s, and 5S rRNA transcripts were calculated using respective qPCR standard curves. cDNA synthesis for the qPCR reactions was performed using 10 ng of indicated RNA samples as input. Experiments were repeated using two biological replicates. Error bars indicate standard deviation.

    Article Snippet: In addition, where indicated, fragment size distribution of RNA or DNA samples was assessed on the Agilent 2100 BioAnalyzer using the Agilent RNA 6000 Pico Kit or Agilent High Sensitivity DNA Kit (Agilent Technologies), respectively.

    Techniques: Derivative Assay, Isolation, Real-time Polymerase Chain Reaction, Standard Deviation

    Experimental infection, parasite isolation, and RNA preparation. A)  Immunofluorescence assay using sheep immune serum against  T. gondii  revealed numerous infected enterocytes. Nuclei are counterstained with DAPI. Shown in the top panel is a 20x magnification of a section of the small intestine (bar = 100 μM) where villi are visible. The bottom panel at 100x magnification shows a schizont containing several merozoites (scale bar = 5 μm).  B)  Enterocytes containing CZ-strain merozoite stages were stripped away selectively, leaving the villus structure and the cells of the  lamina propria  intact. Histology section (Hematoxilin    Eosin stained) showing stripped villi at day 5 post infection.  C)  Microscopic examination of parasites in the detergent washed preparation showed only merozoite stages.  D)  Quality control of total RNA extracted from parasite preparations separated on an Agilent RNA 6000 Pico Chip. The bands generated by host 28S/18S ribosomal RNA (arrowheads) and parasite 26S/18S ribosomal RNA (arrows) as well as a size marker are indicated. The samples analyzed were: raw, unprocessed material from scraped intestinal lining; Tween 80, material that was syringe-passaged and washed twice with PBS/0.05% Tween 80; Percoll, highly enriched parasite fraction after detergent treatment and Percoll gradient centrifugation; Tachy, RNA prepared from tachyzoites grown in cell culture with human foreskin fibroblasts as host cells.

    Journal: BMC Genomics

    Article Title: Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes

    doi: 10.1186/s12864-015-1225-x

    Figure Lengend Snippet: Experimental infection, parasite isolation, and RNA preparation. A) Immunofluorescence assay using sheep immune serum against T. gondii revealed numerous infected enterocytes. Nuclei are counterstained with DAPI. Shown in the top panel is a 20x magnification of a section of the small intestine (bar = 100 μM) where villi are visible. The bottom panel at 100x magnification shows a schizont containing several merozoites (scale bar = 5 μm). B) Enterocytes containing CZ-strain merozoite stages were stripped away selectively, leaving the villus structure and the cells of the lamina propria intact. Histology section (Hematoxilin Eosin stained) showing stripped villi at day 5 post infection. C) Microscopic examination of parasites in the detergent washed preparation showed only merozoite stages. D) Quality control of total RNA extracted from parasite preparations separated on an Agilent RNA 6000 Pico Chip. The bands generated by host 28S/18S ribosomal RNA (arrowheads) and parasite 26S/18S ribosomal RNA (arrows) as well as a size marker are indicated. The samples analyzed were: raw, unprocessed material from scraped intestinal lining; Tween 80, material that was syringe-passaged and washed twice with PBS/0.05% Tween 80; Percoll, highly enriched parasite fraction after detergent treatment and Percoll gradient centrifugation; Tachy, RNA prepared from tachyzoites grown in cell culture with human foreskin fibroblasts as host cells.

    Article Snippet: The quality of the RNA was analysed using the Agilent RNA 6000 Pico Kit (Agilent) and a Bioanalyzer 2100 (Agilent) (Figure D).

    Techniques: Infection, Isolation, Immunofluorescence, Staining, Chromatin Immunoprecipitation, Generated, Marker, Gradient Centrifugation, Cell Culture

    Bioanalyzer electropherogram analysis of fragmentation time-points of leukocyte RNA. Arbitrary fluorescence units (FU) are plotted as a function of RNA size in nucleotides (nt). ( A ) Analysis of fragmentation time-points 15–180 min with Agilent RNA 6000 Pico Kit shows the expected ribosomal RNA peaks in non-fragmented sample (in red), whereas after all fragmentation time-points the majority of RNAs are below 200 nt in size. ( B ) Analysis of fragmentation time-points from 45 to 420 min with Agilent Small RNA Kit shows that the majority of RNAs are smaller than 40 nt in size for all time-points, and that a plateau is reached after approximately 180 min, as no further reduction in size is observed with longer fragmentation times. For the longer fragmentation periods, reduced amounts of RNA (marked with a star) were used to better simulate the enzymatic kinetics in the presence of less (but still detectable) RNA, and the plateau region is still the same.

    Journal: Scientific Reports

    Article Title: A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies

    doi: 10.1038/s41598-017-14264-5

    Figure Lengend Snippet: Bioanalyzer electropherogram analysis of fragmentation time-points of leukocyte RNA. Arbitrary fluorescence units (FU) are plotted as a function of RNA size in nucleotides (nt). ( A ) Analysis of fragmentation time-points 15–180 min with Agilent RNA 6000 Pico Kit shows the expected ribosomal RNA peaks in non-fragmented sample (in red), whereas after all fragmentation time-points the majority of RNAs are below 200 nt in size. ( B ) Analysis of fragmentation time-points from 45 to 420 min with Agilent Small RNA Kit shows that the majority of RNAs are smaller than 40 nt in size for all time-points, and that a plateau is reached after approximately 180 min, as no further reduction in size is observed with longer fragmentation times. For the longer fragmentation periods, reduced amounts of RNA (marked with a star) were used to better simulate the enzymatic kinetics in the presence of less (but still detectable) RNA, and the plateau region is still the same.

    Article Snippet: As EV-derived RNA obtained from plasma cannot be generally visualized in the Bioanalyzer due to its very low amounts, the fragmentation time was optimized with RNA extracted from peripheral blood leukocytes, analyzed with the Agilent RNA 6000 Pico Kit.

    Techniques: Fluorescence

    Total RNA profiles for human and bovine MEV and cream fractions. Total RNA contained in fluff (red) and serum (green) MEV fractions were isolated from SEC F1/S1 peaks concentrated by reverse osmosis. Cream was sampled from the first centrifugation of raw milk. RNA extracts were analysed on Agilent RNA 6000 Pico chips using an Agilent Bioanalyzer. The total MEV RNA content was related to the initial start volume of milk (ng ml –1  milk). The peak at 25 nt is an internal standard. Abbreviations: FU, fluorescence units; nt, nucleotides; hF1, human fluff MEV; hS1, human milk serum MEV; bF1, bovine fluff MEV; bS1, bovine milk serum MEV.

    Journal: Journal of Extracellular Vesicles

    Article Title: Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography

    doi: 10.1080/20013078.2017.1294340

    Figure Lengend Snippet: Total RNA profiles for human and bovine MEV and cream fractions. Total RNA contained in fluff (red) and serum (green) MEV fractions were isolated from SEC F1/S1 peaks concentrated by reverse osmosis. Cream was sampled from the first centrifugation of raw milk. RNA extracts were analysed on Agilent RNA 6000 Pico chips using an Agilent Bioanalyzer. The total MEV RNA content was related to the initial start volume of milk (ng ml –1 milk). The peak at 25 nt is an internal standard. Abbreviations: FU, fluorescence units; nt, nucleotides; hF1, human fluff MEV; hS1, human milk serum MEV; bF1, bovine fluff MEV; bS1, bovine milk serum MEV.

    Article Snippet: Total RNA from −80°C preserved samples, including fluff and milk serum MEV isolates obtained from the same milk lot, was isolated using the miRCURYTM RNA Isolation Kit, and analysed in triplicates on a Agilent RNA 6000 Pico kit chip using a Agilent Bioanalyzer ( ).

    Techniques: Isolation, Size-exclusion Chromatography, Centrifugation, Fluorescence