Structured Review

Agilent technologies bioanalyser
AML-BMSCs have altered gene expression profiles and release exosomes that are enriched in select mi-RNA. (A) Light micrograph and immunophenotype of primary AML-BMSCs and N-BMSCs showing adherent, fibroblastic morphology and positivity for CD90 and negativity for CD45 epitopes. (B) mRNA expression of stromal transcripts C-X-C Chemokine Ligands CXCL12 , CXCL1 and KITLG ( SCF ). (C) Quantitative expression by polymerase chain reaction of select gene transcripts from AML-BMSCs ( n = 13) normalized to N-BMSCs ( n = 4, n = 3 for CXCL12 ). Expression is calculated relative to GAPDH , p-values reflect mean differences between N-BMSCs and AML-BMSCs for the genes shown. (D) Electron micrograph of 30–100 nm sized, cup-shaped vesicles (top left) showing the presence of the exosome markers CD9 and CD81 from both populations (bottom left). Vesicle size and concentration, by percentage of 100 vesicles measured from transmission electron microscope (middle top) and by nanovesicle tracking analysis (middle bottom) from AML-BMSC and N-BMSC. <t>Bioanalyser</t> electropherogram of RNA from stromal cells and their exosomes (right top and bottom), revealing enrichment of small RNA in stromal exosomes. (E) Graphs display the relative abundance of MIR155 and MIR375 in parent stromal cells versus the exosomes released during the same passage number, expressed relative to snRNA U6 . Each cell-exosome pair is derived from a separate patient. ‘Early passage’ cells were used from their second to fourth passage, and ‘late-passage’ cells were used in their 10–12th passage; * P
Bioanalyser, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Alterations in acute myeloid leukaemia bone marrow stromal cell exosome content coincide with gains in tyrosine kinase inhibitor resistance"

Article Title: Alterations in acute myeloid leukaemia bone marrow stromal cell exosome content coincide with gains in tyrosine kinase inhibitor resistance

Journal: British journal of haematology

doi: 10.1111/bjh.13551

AML-BMSCs have altered gene expression profiles and release exosomes that are enriched in select mi-RNA. (A) Light micrograph and immunophenotype of primary AML-BMSCs and N-BMSCs showing adherent, fibroblastic morphology and positivity for CD90 and negativity for CD45 epitopes. (B) mRNA expression of stromal transcripts C-X-C Chemokine Ligands CXCL12 , CXCL1 and KITLG ( SCF ). (C) Quantitative expression by polymerase chain reaction of select gene transcripts from AML-BMSCs ( n = 13) normalized to N-BMSCs ( n = 4, n = 3 for CXCL12 ). Expression is calculated relative to GAPDH , p-values reflect mean differences between N-BMSCs and AML-BMSCs for the genes shown. (D) Electron micrograph of 30–100 nm sized, cup-shaped vesicles (top left) showing the presence of the exosome markers CD9 and CD81 from both populations (bottom left). Vesicle size and concentration, by percentage of 100 vesicles measured from transmission electron microscope (middle top) and by nanovesicle tracking analysis (middle bottom) from AML-BMSC and N-BMSC. Bioanalyser electropherogram of RNA from stromal cells and their exosomes (right top and bottom), revealing enrichment of small RNA in stromal exosomes. (E) Graphs display the relative abundance of MIR155 and MIR375 in parent stromal cells versus the exosomes released during the same passage number, expressed relative to snRNA U6 . Each cell-exosome pair is derived from a separate patient. ‘Early passage’ cells were used from their second to fourth passage, and ‘late-passage’ cells were used in their 10–12th passage; * P
Figure Legend Snippet: AML-BMSCs have altered gene expression profiles and release exosomes that are enriched in select mi-RNA. (A) Light micrograph and immunophenotype of primary AML-BMSCs and N-BMSCs showing adherent, fibroblastic morphology and positivity for CD90 and negativity for CD45 epitopes. (B) mRNA expression of stromal transcripts C-X-C Chemokine Ligands CXCL12 , CXCL1 and KITLG ( SCF ). (C) Quantitative expression by polymerase chain reaction of select gene transcripts from AML-BMSCs ( n = 13) normalized to N-BMSCs ( n = 4, n = 3 for CXCL12 ). Expression is calculated relative to GAPDH , p-values reflect mean differences between N-BMSCs and AML-BMSCs for the genes shown. (D) Electron micrograph of 30–100 nm sized, cup-shaped vesicles (top left) showing the presence of the exosome markers CD9 and CD81 from both populations (bottom left). Vesicle size and concentration, by percentage of 100 vesicles measured from transmission electron microscope (middle top) and by nanovesicle tracking analysis (middle bottom) from AML-BMSC and N-BMSC. Bioanalyser electropherogram of RNA from stromal cells and their exosomes (right top and bottom), revealing enrichment of small RNA in stromal exosomes. (E) Graphs display the relative abundance of MIR155 and MIR375 in parent stromal cells versus the exosomes released during the same passage number, expressed relative to snRNA U6 . Each cell-exosome pair is derived from a separate patient. ‘Early passage’ cells were used from their second to fourth passage, and ‘late-passage’ cells were used in their 10–12th passage; * P

Techniques Used: Expressing, Polymerase Chain Reaction, Concentration Assay, Transmission Assay, Microscopy, Derivative Assay

2) Product Images from "Accelerated Dosing Frequency of a Pulmonary Formulation of Tissue Plasminogen Activator is Well-Tolerated in Mice"

Article Title: Accelerated Dosing Frequency of a Pulmonary Formulation of Tissue Plasminogen Activator is Well-Tolerated in Mice

Journal:

doi: 10.1111/j.1440-1681.2008.05011.x

Representative electropherograms of bioanalyser data of a pulmonary formulation of mouse tissue plasminogen activator (pf-mtPA) and interactions between pf-mtPA and a stable mutant form of human plasminogen activator inhibitor-1 (CPAI) at pH 7.3. (a)
Figure Legend Snippet: Representative electropherograms of bioanalyser data of a pulmonary formulation of mouse tissue plasminogen activator (pf-mtPA) and interactions between pf-mtPA and a stable mutant form of human plasminogen activator inhibitor-1 (CPAI) at pH 7.3. (a)

Techniques Used: Mutagenesis

3) Product Images from "Quality control on the frontier"

Article Title: Quality control on the frontier

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2014.00157

Library fragment size distribution . Bioanalyser fluorescence values (black), realignment of paired end reads against reference genome or de novo assembly and adjusted by 126 bases to account for adapters (red) for libraries with average sizes of 360 bases (A) , 550 bases (B) , and 810 bases (C) .
Figure Legend Snippet: Library fragment size distribution . Bioanalyser fluorescence values (black), realignment of paired end reads against reference genome or de novo assembly and adjusted by 126 bases to account for adapters (red) for libraries with average sizes of 360 bases (A) , 550 bases (B) , and 810 bases (C) .

Techniques Used: Fluorescence

Examples of Bioanalyser assay interpretation for a variety of RNAs. (A) Standard Eukaryotic RNA shows a 28S rRNA band at 4.5 kb that should be twice the intensity of the 18S rRNA band at 1.9 kb (human) resulting in a RIN = 8.0–10.0. Small peaks are sometimes present after the marker that represent 5S and 5.8S subunits, tRNAs and small RNA fragments about 100 bp; these are more obvious when using phenol or trizol exterection methods, QIagen columns will generally remove small RNAs. When degraded 28S RNA is reduced and more fragments are detected around the 18S RNA subunit resulting in RIN = 6.4, which is below the quality required for high throughput DNA sequencing. Invertebrate RNA results in fragmentation of the 28S rRNA into two bands that co-migrate with the 18S rRNA resulting in aberrant RIN score of
Figure Legend Snippet: Examples of Bioanalyser assay interpretation for a variety of RNAs. (A) Standard Eukaryotic RNA shows a 28S rRNA band at 4.5 kb that should be twice the intensity of the 18S rRNA band at 1.9 kb (human) resulting in a RIN = 8.0–10.0. Small peaks are sometimes present after the marker that represent 5S and 5.8S subunits, tRNAs and small RNA fragments about 100 bp; these are more obvious when using phenol or trizol exterection methods, QIagen columns will generally remove small RNAs. When degraded 28S RNA is reduced and more fragments are detected around the 18S RNA subunit resulting in RIN = 6.4, which is below the quality required for high throughput DNA sequencing. Invertebrate RNA results in fragmentation of the 28S rRNA into two bands that co-migrate with the 18S rRNA resulting in aberrant RIN score of

Techniques Used: Marker, High Throughput Screening Assay, DNA Sequencing

4) Product Images from "Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood"

Article Title: Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood

Journal: Journal of Extracellular Vesicles

doi: 10.3402/jev.v3.23743

Small RNA profiles extracted from intracellular, cell-free and exosomal isolation from blood before and after RNaseA treatment. RNA was extracted from samples and run on a Small RNA Bioanalyser assay. Experiments shown here are representative of samples collected from 1 volunteer.
Figure Legend Snippet: Small RNA profiles extracted from intracellular, cell-free and exosomal isolation from blood before and after RNaseA treatment. RNA was extracted from samples and run on a Small RNA Bioanalyser assay. Experiments shown here are representative of samples collected from 1 volunteer.

Techniques Used: Isolation

Related Articles

Electrophoresis:

Article Title: Quality control on the frontier
Article Snippet: .. Assays that use fluorescent dyes in conjunction with microfluidic electrophoresis include Bioanalyser (Agilent), Labchip GX (Perkin Elmer), QIAxcel (QIAGEN), and Fragment Analyser (VH bio ltd); these instruments can be used to analyse dsDNA fragments, RNA or prepared NGS libraries where material is precious. .. UV absorbance ratios at 230:260 nm and 260:280 nm can provide additional information regarding purity of the sample, in particular, presence of phenol which absorbs with a peak at 270 nm, can contribute to the over-estimation of DNA concentration, whereas, humic acids that may be present in DNA isolated from soil absorb at 230 nm, as do phelolate ions and thiocyanates that may be used to isolate RNA.

Next-Generation Sequencing:

Article Title: Quality control on the frontier
Article Snippet: .. Assays that use fluorescent dyes in conjunction with microfluidic electrophoresis include Bioanalyser (Agilent), Labchip GX (Perkin Elmer), QIAxcel (QIAGEN), and Fragment Analyser (VH bio ltd); these instruments can be used to analyse dsDNA fragments, RNA or prepared NGS libraries where material is precious. .. UV absorbance ratios at 230:260 nm and 260:280 nm can provide additional information regarding purity of the sample, in particular, presence of phenol which absorbs with a peak at 270 nm, can contribute to the over-estimation of DNA concentration, whereas, humic acids that may be present in DNA isolated from soil absorb at 230 nm, as do phelolate ions and thiocyanates that may be used to isolate RNA.

Purification:

Article Title: Subgroup 4 R2R3-MYBs in conifer trees: gene family expansion and contribution to the isoprenoid- and flavonoid-oriented responses
Article Snippet: .. Standard curves were based on dilution series covering five orders of magnitude (10−1 –10−6 ng μl−1 ) prepared for each cDNA linearized with Eco RI or Bam HI, purified on Qiaquick columns (Qiagen), and verified on a bioAnalyser (model 2100, DNA 1000 LabChip kit, Agilent Technologies). ..

Real-time Polymerase Chain Reaction:

Article Title: Insectivorous bats selectively source moths and eat mostly pest insects on dryland and irrigated cotton farms. Insectivorous bats selectively source moths and eat mostly pest insects on dryland and irrigated cotton farms
Article Snippet: .. The equimolar pool was then measured by qPCR (KAPA, Roche) and visualized on the Bioanalyser (Agilent). .. Sequencing was completed by the AGRF Melbourne Node on the Illumina MiSeq with 2 × 150‐bp paired‐end v2 chemistry.

Incubation:

Article Title: Accelerated Dosing Frequency of a Pulmonary Formulation of Tissue Plasminogen Activator is Well-Tolerated in Mice
Article Snippet: .. Mouse tPA protein (5 µg) was incubated in the presence or absence of CPAI (15 µg) in Tris-buffered saline (TBS; ×1) at room temperature for 1 min to 1 h. Each reaction was stopped by the addition of bioanalyser sample buffer (Agilent Technologies, Santa Clara, CA, USA) and heating to 70°C for 10 min. Each denatured sample was then diluted to a final volume of 90 µL with water. .. Samples were assayed and analysed by an Agilent 2100 bioanalyser according to the Protein 200 Plus assay protocol.

Sequencing:

Article Title: Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture
Article Snippet: .. ATAC libraries were quantified using the BioAnalyser (Agilent) and pooled to 4 nM for sequencing on the HiSeq 2500 or HiSeq 4000 system (Illumina). ..

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    Agilent technologies agilent 2100 bioanalyser
    Isolation of cotton tissues from paraffin-embedded sections by laser microdissection (LMD). Isolation of cotton tissues from paraffin-embedded sections by laser microdissection (LMD). Sections before LMD (a, c, e) , and sections after LM (b, d, f) . The area selected for laser microdissection is outlined in green (a region near the damage caused by larvae feeding, which comprised the stamen tissue, viewed at a , c and d ) or blue (a region farther from the injured area, which comprised the carpel tissue, viewed at a , e , and f ). The assessment of extracted RNA integrity from the stamen and carpel are shown in g and h , respectively. Electropherograms were obtained with an <t>Agilent</t> 2100 <t>Bioanalyser.</t> Open and closed arrowheads indicate the 18S and 28S ribosomal RNA peaks, respectively. RNA quality is expressed as the RNA integrity number (RIN). Scale bars = 100 μm.
    Agilent 2100 Bioanalyser, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 655 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Isolation of cotton tissues from paraffin-embedded sections by laser microdissection (LMD). Isolation of cotton tissues from paraffin-embedded sections by laser microdissection (LMD). Sections before LMD (a, c, e) , and sections after LM (b, d, f) . The area selected for laser microdissection is outlined in green (a region near the damage caused by larvae feeding, which comprised the stamen tissue, viewed at a , c and d ) or blue (a region farther from the injured area, which comprised the carpel tissue, viewed at a , e , and f ). The assessment of extracted RNA integrity from the stamen and carpel are shown in g and h , respectively. Electropherograms were obtained with an Agilent 2100 Bioanalyser. Open and closed arrowheads indicate the 18S and 28S ribosomal RNA peaks, respectively. RNA quality is expressed as the RNA integrity number (RIN). Scale bars = 100 μm.

    Journal: BMC Genomics

    Article Title: Transcriptome analysis of Gossypium hirsutum flower buds infested by cotton boll weevil (Anthonomus grandis) larvae

    doi: 10.1186/1471-2164-15-854

    Figure Lengend Snippet: Isolation of cotton tissues from paraffin-embedded sections by laser microdissection (LMD). Isolation of cotton tissues from paraffin-embedded sections by laser microdissection (LMD). Sections before LMD (a, c, e) , and sections after LM (b, d, f) . The area selected for laser microdissection is outlined in green (a region near the damage caused by larvae feeding, which comprised the stamen tissue, viewed at a , c and d ) or blue (a region farther from the injured area, which comprised the carpel tissue, viewed at a , e , and f ). The assessment of extracted RNA integrity from the stamen and carpel are shown in g and h , respectively. Electropherograms were obtained with an Agilent 2100 Bioanalyser. Open and closed arrowheads indicate the 18S and 28S ribosomal RNA peaks, respectively. RNA quality is expressed as the RNA integrity number (RIN). Scale bars = 100 μm.

    Article Snippet: The quality of total RNA extracted from LMD-collected tissues was assessed using an RNA 6000 Pico kit on the Agilent 2100 Bioanalyser (Agilent Technologies).

    Techniques: Isolation, Laser Capture Microdissection

    AML-BMSCs have altered gene expression profiles and release exosomes that are enriched in select mi-RNA. (A) Light micrograph and immunophenotype of primary AML-BMSCs and N-BMSCs showing adherent, fibroblastic morphology and positivity for CD90 and negativity for CD45 epitopes. (B) mRNA expression of stromal transcripts C-X-C Chemokine Ligands CXCL12 , CXCL1 and KITLG ( SCF ). (C) Quantitative expression by polymerase chain reaction of select gene transcripts from AML-BMSCs ( n = 13) normalized to N-BMSCs ( n = 4, n = 3 for CXCL12 ). Expression is calculated relative to GAPDH , p-values reflect mean differences between N-BMSCs and AML-BMSCs for the genes shown. (D) Electron micrograph of 30–100 nm sized, cup-shaped vesicles (top left) showing the presence of the exosome markers CD9 and CD81 from both populations (bottom left). Vesicle size and concentration, by percentage of 100 vesicles measured from transmission electron microscope (middle top) and by nanovesicle tracking analysis (middle bottom) from AML-BMSC and N-BMSC. Bioanalyser electropherogram of RNA from stromal cells and their exosomes (right top and bottom), revealing enrichment of small RNA in stromal exosomes. (E) Graphs display the relative abundance of MIR155 and MIR375 in parent stromal cells versus the exosomes released during the same passage number, expressed relative to snRNA U6 . Each cell-exosome pair is derived from a separate patient. ‘Early passage’ cells were used from their second to fourth passage, and ‘late-passage’ cells were used in their 10–12th passage; * P

    Journal: British journal of haematology

    Article Title: Alterations in acute myeloid leukaemia bone marrow stromal cell exosome content coincide with gains in tyrosine kinase inhibitor resistance

    doi: 10.1111/bjh.13551

    Figure Lengend Snippet: AML-BMSCs have altered gene expression profiles and release exosomes that are enriched in select mi-RNA. (A) Light micrograph and immunophenotype of primary AML-BMSCs and N-BMSCs showing adherent, fibroblastic morphology and positivity for CD90 and negativity for CD45 epitopes. (B) mRNA expression of stromal transcripts C-X-C Chemokine Ligands CXCL12 , CXCL1 and KITLG ( SCF ). (C) Quantitative expression by polymerase chain reaction of select gene transcripts from AML-BMSCs ( n = 13) normalized to N-BMSCs ( n = 4, n = 3 for CXCL12 ). Expression is calculated relative to GAPDH , p-values reflect mean differences between N-BMSCs and AML-BMSCs for the genes shown. (D) Electron micrograph of 30–100 nm sized, cup-shaped vesicles (top left) showing the presence of the exosome markers CD9 and CD81 from both populations (bottom left). Vesicle size and concentration, by percentage of 100 vesicles measured from transmission electron microscope (middle top) and by nanovesicle tracking analysis (middle bottom) from AML-BMSC and N-BMSC. Bioanalyser electropherogram of RNA from stromal cells and their exosomes (right top and bottom), revealing enrichment of small RNA in stromal exosomes. (E) Graphs display the relative abundance of MIR155 and MIR375 in parent stromal cells versus the exosomes released during the same passage number, expressed relative to snRNA U6 . Each cell-exosome pair is derived from a separate patient. ‘Early passage’ cells were used from their second to fourth passage, and ‘late-passage’ cells were used in their 10–12th passage; * P

    Article Snippet: We determined the spectrum of BMSC exosome RNA using a Bioanalyser (Agilent, Santa Clara, CA, USA) and observed a relatively greater abundance of small RNAs compared with the parent cell ( ).

    Techniques: Expressing, Polymerase Chain Reaction, Concentration Assay, Transmission Assay, Microscopy, Derivative Assay

    Representative electropherograms of bioanalyser data of a pulmonary formulation of mouse tissue plasminogen activator (pf-mtPA) and interactions between pf-mtPA and a stable mutant form of human plasminogen activator inhibitor-1 (CPAI) at pH 7.3. (a)

    Journal:

    Article Title: Accelerated Dosing Frequency of a Pulmonary Formulation of Tissue Plasminogen Activator is Well-Tolerated in Mice

    doi: 10.1111/j.1440-1681.2008.05011.x

    Figure Lengend Snippet: Representative electropherograms of bioanalyser data of a pulmonary formulation of mouse tissue plasminogen activator (pf-mtPA) and interactions between pf-mtPA and a stable mutant form of human plasminogen activator inhibitor-1 (CPAI) at pH 7.3. (a)

    Article Snippet: Mouse tPA protein (5 µg) was incubated in the presence or absence of CPAI (15 µg) in Tris-buffered saline (TBS; ×1) at room temperature for 1 min to 1 h. Each reaction was stopped by the addition of bioanalyser sample buffer (Agilent Technologies, Santa Clara, CA, USA) and heating to 70°C for 10 min. Each denatured sample was then diluted to a final volume of 90 µL with water.

    Techniques: Mutagenesis

    Xbp1 mRNA cleavage by human Ire1α. ( A ) Elution profiles from Agilent Bioanalyser of (left) untreated Xbp1 mRNA, (middle) Xbp1 mRNA incubated with dephosphorylated Ire1α, and (right) Xbp1 mRNA incubated with phosphorylated Ire1α. Reaction conditions are given in Materials and methods section. The peak eluting at a position corresponding to 800 nucleotides in the left and middle panels is the unspliced Xbp1 mRNA, whereas the pair of peaks eluting at 300 and 500 nucleotides, respectively, in the right panel, corresponds to the two cleaved products of Xbp1 mRNA. The peak at 25 nucleotides in all three panels is a calibration marker. ( B ) In vitro cleavage activity of Ire1α, wild-type, and Q636A mutant, on a fluorescently tagged oligonucleotide encapsulating the sequence and secondary structure of a known Ire1α cleavage site in human Xbp1 mRNA. Cleavage generates a fluorescent species with higher gel mobility. The Q636A mutation, which causes a defect in autophosphorylation, results in a similar decrease in RNase activity, confirming the dependence of RNase activation on autophosphorylation. ( C ) Sunitinib inhibits Xbp1 mRNA splicing in vivo . Myeloma cell lines (U266+H929) were treated with tunicamycin to induce ER stress, in the presence or absence of sunitinib, and levels of spliced (Xbp1s) and unspliced (Xbp1u) mRNAs in the treated cells were determined by quantitative real-time PCR using LUX primers (see Materials and methods), and plotted as relative Xbp1u:Xbp1s ratio. In both cell lines, the addition of the Ire1α kinase inhibitor sunitinib significantly inhibits the splicing of Xbp1 mRNA. ( D ) Similar results were obtained by RT–PCR amplification. ( E ) Western blot of protein extracts from cells treated as in ( C ), showing inhibition of Xbp1 protein production in ER-stressed cells treated with sunitinib. Actin is shown as a loading control.

    Journal: The EMBO Journal

    Article Title: Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response

    doi: 10.1038/emboj.2011.18

    Figure Lengend Snippet: Xbp1 mRNA cleavage by human Ire1α. ( A ) Elution profiles from Agilent Bioanalyser of (left) untreated Xbp1 mRNA, (middle) Xbp1 mRNA incubated with dephosphorylated Ire1α, and (right) Xbp1 mRNA incubated with phosphorylated Ire1α. Reaction conditions are given in Materials and methods section. The peak eluting at a position corresponding to 800 nucleotides in the left and middle panels is the unspliced Xbp1 mRNA, whereas the pair of peaks eluting at 300 and 500 nucleotides, respectively, in the right panel, corresponds to the two cleaved products of Xbp1 mRNA. The peak at 25 nucleotides in all three panels is a calibration marker. ( B ) In vitro cleavage activity of Ire1α, wild-type, and Q636A mutant, on a fluorescently tagged oligonucleotide encapsulating the sequence and secondary structure of a known Ire1α cleavage site in human Xbp1 mRNA. Cleavage generates a fluorescent species with higher gel mobility. The Q636A mutation, which causes a defect in autophosphorylation, results in a similar decrease in RNase activity, confirming the dependence of RNase activation on autophosphorylation. ( C ) Sunitinib inhibits Xbp1 mRNA splicing in vivo . Myeloma cell lines (U266+H929) were treated with tunicamycin to induce ER stress, in the presence or absence of sunitinib, and levels of spliced (Xbp1s) and unspliced (Xbp1u) mRNAs in the treated cells were determined by quantitative real-time PCR using LUX primers (see Materials and methods), and plotted as relative Xbp1u:Xbp1s ratio. In both cell lines, the addition of the Ire1α kinase inhibitor sunitinib significantly inhibits the splicing of Xbp1 mRNA. ( D ) Similar results were obtained by RT–PCR amplification. ( E ) Western blot of protein extracts from cells treated as in ( C ), showing inhibition of Xbp1 protein production in ER-stressed cells treated with sunitinib. Actin is shown as a loading control.

    Article Snippet: In all, 150 ng Ire1α was incubated with 500 ng Xbp1u mRNA at 37°C for 90 min, purified (GeneChip IVT cRNA Cleanup Kit) and analysed using the Agilent Bioanalyser system (Agilent Technologies) according to the manufacturer's instructions.

    Techniques: Incubation, Marker, In Vitro, Activity Assay, Mutagenesis, Sequencing, Activation Assay, In Vivo, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Inhibition