Structured Review

TaKaRa cdna template
IRF4, <t>vIRF3,</t> and BATF cooperatively promote IRF4-SE activity. (A) A 500-bp sequence centered on the prominent IRF4 and vIRF3 ChIP-Seq peaks ∼63 kb upstream of the IRF4 TSS drove eGFP expression from a lentiviral enhancer reporter (IRF4SE-eGFP) after transduction into PEL cell lines BC-3, BCBL-1, and BC-1 but not the IRF4/vIRF3-negative cell lines 293 and BJAB. Cell lines were transduced at MOI 3 and analyzed 4 days after transduction. Similar reporters containing the SV40 enhancer or a minimal promoter (mP) served as positive or negative controls, respectively. Reporters were titrated by qRT-PCR and, where possible, FACS analysis prior to transduction. Data are representative of results from n = 3 biological replicates. (B) The lentiviral IRF4SE or mP eGFP reporters were transduced into 293 cells at MOI 3, together with the indicated combinations of lentiviral expression vectors for vIRF3, IRF4, or BATF. Expression of mCherry served as a negative control. eGFP mean fluorescence intensities were measured by FACS analysis on day 3 after transduction and are shown relative to values from cells that were not transduced with lentiviral <t>cDNA</t> expression vectors. Error bars represent SEM from 3 (mP) or 4 (IRF4SE) biological replicates. *, P
Cdna Template, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 298 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Kaposi’s Sarcoma-Associated Herpesvirus Drives a Super-Enhancer-Mediated Survival Gene Expression Program in Primary Effusion Lymphoma"

Article Title: Kaposi’s Sarcoma-Associated Herpesvirus Drives a Super-Enhancer-Mediated Survival Gene Expression Program in Primary Effusion Lymphoma

Journal: mBio

doi: 10.1128/mBio.01457-20

IRF4, vIRF3, and BATF cooperatively promote IRF4-SE activity. (A) A 500-bp sequence centered on the prominent IRF4 and vIRF3 ChIP-Seq peaks ∼63 kb upstream of the IRF4 TSS drove eGFP expression from a lentiviral enhancer reporter (IRF4SE-eGFP) after transduction into PEL cell lines BC-3, BCBL-1, and BC-1 but not the IRF4/vIRF3-negative cell lines 293 and BJAB. Cell lines were transduced at MOI 3 and analyzed 4 days after transduction. Similar reporters containing the SV40 enhancer or a minimal promoter (mP) served as positive or negative controls, respectively. Reporters were titrated by qRT-PCR and, where possible, FACS analysis prior to transduction. Data are representative of results from n = 3 biological replicates. (B) The lentiviral IRF4SE or mP eGFP reporters were transduced into 293 cells at MOI 3, together with the indicated combinations of lentiviral expression vectors for vIRF3, IRF4, or BATF. Expression of mCherry served as a negative control. eGFP mean fluorescence intensities were measured by FACS analysis on day 3 after transduction and are shown relative to values from cells that were not transduced with lentiviral cDNA expression vectors. Error bars represent SEM from 3 (mP) or 4 (IRF4SE) biological replicates. *, P
Figure Legend Snippet: IRF4, vIRF3, and BATF cooperatively promote IRF4-SE activity. (A) A 500-bp sequence centered on the prominent IRF4 and vIRF3 ChIP-Seq peaks ∼63 kb upstream of the IRF4 TSS drove eGFP expression from a lentiviral enhancer reporter (IRF4SE-eGFP) after transduction into PEL cell lines BC-3, BCBL-1, and BC-1 but not the IRF4/vIRF3-negative cell lines 293 and BJAB. Cell lines were transduced at MOI 3 and analyzed 4 days after transduction. Similar reporters containing the SV40 enhancer or a minimal promoter (mP) served as positive or negative controls, respectively. Reporters were titrated by qRT-PCR and, where possible, FACS analysis prior to transduction. Data are representative of results from n = 3 biological replicates. (B) The lentiviral IRF4SE or mP eGFP reporters were transduced into 293 cells at MOI 3, together with the indicated combinations of lentiviral expression vectors for vIRF3, IRF4, or BATF. Expression of mCherry served as a negative control. eGFP mean fluorescence intensities were measured by FACS analysis on day 3 after transduction and are shown relative to values from cells that were not transduced with lentiviral cDNA expression vectors. Error bars represent SEM from 3 (mP) or 4 (IRF4SE) biological replicates. *, P

Techniques Used: Activity Assay, Sequencing, Chromatin Immunoprecipitation, Expressing, Transduction, Quantitative RT-PCR, FACS, Negative Control, Fluorescence

Related Articles

Clone Assay:

Article Title: Kaposi’s Sarcoma-Associated Herpesvirus Drives a Super-Enhancer-Mediated Survival Gene Expression Program in Primary Effusion Lymphoma
Article Snippet: .. The vIRF3 cDNA was amplified from a cDNA template that was originally amplified from BC-1 genomic DNA and initially cloned into pMSCV-hyg (Clontech), using primers 4203 and 4204. .. The 3× FLAG tag was amplified from pTRIPZ/KapA-3XFLAG ( ) using primers 4205 and 4141.

Amplification:

Article Title: Kaposi’s Sarcoma-Associated Herpesvirus Drives a Super-Enhancer-Mediated Survival Gene Expression Program in Primary Effusion Lymphoma
Article Snippet: .. The vIRF3 cDNA was amplified from a cDNA template that was originally amplified from BC-1 genomic DNA and initially cloned into pMSCV-hyg (Clontech), using primers 4203 and 4204. .. The 3× FLAG tag was amplified from pTRIPZ/KapA-3XFLAG ( ) using primers 4205 and 4141.

Article Title: Deep sequence analysis of HIV adaptation following vertical transmission reveals the impact of immune pressure on the evolution of HIV
Article Snippet: .. An upper limit of 20,000 RNA copies was utilized to allow each cDNA template to contain a unique PID sequence. cDNA was purified twice to remove any unbound PIDs before PCR amplification with a sample specific primer and a generic primer for the nonspecific region using PrimeSTAR GXL DNA Polymerase (Takara Bio Inc., Japan). .. First round products were purified using the MinElute PCR Purification kit (QIAGEN), as per the manufacturer’s instructions.

Article Title: Clinical significance of down-regulated HINT2 in hepatocellular carcinoma
Article Snippet: .. Amplification reactions containing 1 μl cDNA template, 0.3 μl of the forward and reverse primer each (10 μM), 0.2 μl 50 × ROX Reference Dye II (Takara) and 5 μl 2 × SYBR Premix Dimer Eraser were mixed and brought to a total volume of 10 μl. ..

Article Title: Long non-coding RNA ANRIL promotes tumorgenesis through regulation of FGFR1 expression by sponging miR-125a-3p in head and neck squamous cell carcinoma
Article Snippet: .. The cDNA template was amplified by real-time RT-PCR using the SYBR Premix Dimmer Eraser kit (TaKaRa). .. Gene expression in each sample was normalized to the GADPH expression.

Article Title: Long-Range Polymerase Chain Reaction Method for Sequencing the Ebola Virus Genome From Ecological and Clinical Samples
Article Snippet: .. Each 50 µL LRPCR master mix contained 0.2 µM of each primer (first round of PCR, Amplicon 1: 5′-GATCTTTTGTGTGCGAATAACTATGAGGA-3′ and 5′-TGCAATAGGGCCATTCCTTTGT-3′, Amplicon 2: 5′-GAA TATCGCTCTCCAGTTACCGTGT-3′ and 5′-GACCATTTTT CCAGGGATCCTTTT-3′; second round of PCR, Amplicon 1: 5′-GATCTTTTGTGTGCGAATAACTATGAGGA-3′ and 5′-CACATCAAACTCAATACCAGCCCA-3′, Amplicon 2: 5′-TGCTGTCGTTGTTTCAGGGTTAA-3′ and 5′-GACCA TTTTTCCAGGGATCCTTTT-3′), 1 × PrimeSTAR GXL Buffer, 200 µM each deoxynucleotide triphosphate (dNTP), 5 µL of cDNA template, 1.25 Units of PrimeSTAR GXL DNA Polymerase (Takara Bio USA). .. The LRPCR mixture was incubated at 98°C for 2 minutes for the initial denaturation, followed by 4 cycles at 98°C for 10 seconds, 68°C for 15 seconds (−2°C per cycle), 72°C for 10 minutes, before an additional 26 cycles of 98°C for 10 seconds, 58°C for 15 seconds, 72°C for 10 minutes.

Isolation:

Article Title: Characterizing and evaluating the expression of the type IIb sodium-dependent phosphate cotransporter (slc34a2) gene and its potential influence on phosphorus utilization efficiency in yellow catfish (Pelteobagrus fulvidraco).
Article Snippet: .. A sodium-dependent phosphate cotransporter gene, NaPi-IIb (slc34a2), was isolated from yellow catfish (Pelteobagrus fulvidraco) intestine through homology cloning and the rapid amplification of cDNA ends. .. A sodium-dependent phosphate cotransporter gene, NaPi-IIb (slc34a2), was isolated from yellow catfish (Pelteobagrus fulvidraco) intestine through homology cloning and the rapid amplification of cDNA ends.

Sequencing:

Article Title: Characterizing and evaluating the expression of the type IIb sodium-dependent phosphate cotransporter (slc34a2) gene and its potential influence on phosphorus utilization efficiency in yellow catfish (Pelteobagrus fulvidraco).
Article Snippet: .. A sodium-dependent phosphate cotransporter gene, NaPi-IIb (slc34a2), was isolated from yellow catfish (Pelteobagrus fulvidraco) intestine through homology cloning and the rapid amplification of cDNA ends. .. A sodium-dependent phosphate cotransporter gene, NaPi-IIb (slc34a2), was isolated from yellow catfish (Pelteobagrus fulvidraco) intestine through homology cloning and the rapid amplification of cDNA ends.

Article Title: Deep sequence analysis of HIV adaptation following vertical transmission reveals the impact of immune pressure on the evolution of HIV
Article Snippet: .. An upper limit of 20,000 RNA copies was utilized to allow each cDNA template to contain a unique PID sequence. cDNA was purified twice to remove any unbound PIDs before PCR amplification with a sample specific primer and a generic primer for the nonspecific region using PrimeSTAR GXL DNA Polymerase (Takara Bio Inc., Japan). .. First round products were purified using the MinElute PCR Purification kit (QIAGEN), as per the manufacturer’s instructions.

Quantitative RT-PCR:

Article Title: Identification of the interaction between vimentin and nucleocapsid protein of transmissible gastroenteritis virus
Article Snippet: .. Real time RT-PCR was performed using a LightCycler 480 II (Roche) in a total volume of 25 μL containing 10 ng of cDNA template, 1× SYBR® Premix Ex Taq™ II (Perfect Real Time, TaKaRa), and a 0.4 μM concentration of each primer. .. After initial denaturation at 95 °C for 2 min, the amplification was performed for 40 cycles, each consisting of denaturation at 95 °C for 5 s and primer annealing at 60 °C for 30 s. Melting curves were obtained, and quantitative analysis of the data was performed in a relative quantification (2−ΔΔCT ) study model.

Article Title: Long non-coding RNA ANRIL promotes tumorgenesis through regulation of FGFR1 expression by sponging miR-125a-3p in head and neck squamous cell carcinoma
Article Snippet: .. The cDNA template was amplified by real-time RT-PCR using the SYBR Premix Dimmer Eraser kit (TaKaRa). .. Gene expression in each sample was normalized to the GADPH expression.

Purification:

Article Title: Deep sequence analysis of HIV adaptation following vertical transmission reveals the impact of immune pressure on the evolution of HIV
Article Snippet: .. An upper limit of 20,000 RNA copies was utilized to allow each cDNA template to contain a unique PID sequence. cDNA was purified twice to remove any unbound PIDs before PCR amplification with a sample specific primer and a generic primer for the nonspecific region using PrimeSTAR GXL DNA Polymerase (Takara Bio Inc., Japan). .. First round products were purified using the MinElute PCR Purification kit (QIAGEN), as per the manufacturer’s instructions.

Real-time Polymerase Chain Reaction:

Article Title: Variation of Growth-to-Ripening Time Interval Induced by Abscisic Acid and Synthetic Auxin affecting Transcriptome and Flavor Compounds in Cabernet Sauvignon Grape Berry
Article Snippet: .. Each real-time PCR reaction (20 μL) contained 2.0 μL of the cDNA template, 1.6 μL of the primer mixture (10 μM each, mixed with equivalent volume), 10.0 μL of 2×SYBR Premix Ex Taq II and 0.4 L of 50× ROX Reference Dye (Takara, Otsu, Japan), and 6.0 μL ddH2O. ..

Concentration Assay:

Article Title: Identification of the interaction between vimentin and nucleocapsid protein of transmissible gastroenteritis virus
Article Snippet: .. Real time RT-PCR was performed using a LightCycler 480 II (Roche) in a total volume of 25 μL containing 10 ng of cDNA template, 1× SYBR® Premix Ex Taq™ II (Perfect Real Time, TaKaRa), and a 0.4 μM concentration of each primer. .. After initial denaturation at 95 °C for 2 min, the amplification was performed for 40 cycles, each consisting of denaturation at 95 °C for 5 s and primer annealing at 60 °C for 30 s. Melting curves were obtained, and quantitative analysis of the data was performed in a relative quantification (2−ΔΔCT ) study model.

Polymerase Chain Reaction:

Article Title: Deep sequence analysis of HIV adaptation following vertical transmission reveals the impact of immune pressure on the evolution of HIV
Article Snippet: .. An upper limit of 20,000 RNA copies was utilized to allow each cDNA template to contain a unique PID sequence. cDNA was purified twice to remove any unbound PIDs before PCR amplification with a sample specific primer and a generic primer for the nonspecific region using PrimeSTAR GXL DNA Polymerase (Takara Bio Inc., Japan). .. First round products were purified using the MinElute PCR Purification kit (QIAGEN), as per the manufacturer’s instructions.

Article Title: Long-Range Polymerase Chain Reaction Method for Sequencing the Ebola Virus Genome From Ecological and Clinical Samples
Article Snippet: .. Each 50 µL LRPCR master mix contained 0.2 µM of each primer (first round of PCR, Amplicon 1: 5′-GATCTTTTGTGTGCGAATAACTATGAGGA-3′ and 5′-TGCAATAGGGCCATTCCTTTGT-3′, Amplicon 2: 5′-GAA TATCGCTCTCCAGTTACCGTGT-3′ and 5′-GACCATTTTT CCAGGGATCCTTTT-3′; second round of PCR, Amplicon 1: 5′-GATCTTTTGTGTGCGAATAACTATGAGGA-3′ and 5′-CACATCAAACTCAATACCAGCCCA-3′, Amplicon 2: 5′-TGCTGTCGTTGTTTCAGGGTTAA-3′ and 5′-GACCA TTTTTCCAGGGATCCTTTT-3′), 1 × PrimeSTAR GXL Buffer, 200 µM each deoxynucleotide triphosphate (dNTP), 5 µL of cDNA template, 1.25 Units of PrimeSTAR GXL DNA Polymerase (Takara Bio USA). .. The LRPCR mixture was incubated at 98°C for 2 minutes for the initial denaturation, followed by 4 cycles at 98°C for 10 seconds, 68°C for 15 seconds (−2°C per cycle), 72°C for 10 minutes, before an additional 26 cycles of 98°C for 10 seconds, 58°C for 15 seconds, 72°C for 10 minutes.

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  • 85
    TaKaRa tissue cdna templates
    <t>RNA</t> isolation, <t>cDNA</t> synthesis, and real-time PCR quantification
    Tissue Cdna Templates, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa mouse brain cdna template
    Tissue distribution of the mouse and monkey UT receptor: (a) Tissue distribution of mouse UT receptor <t>cDNA</t> transcripts by RT – <t>PCR</t> revealed expression within cardiac and vascular (thoracic but not abdominal aorta) tissue in addition to bladder and pancreas. Trace levels of expression are also observed in skeletal muscle, oesophagus, lung and adipose tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (Lower panel) by Southern analysis using full-length UT receptor cDNA probe. (b) Tissue distributions of monkey UT receptor cDNA transcripts by RT – PCR revealed expression within heart (ventricle > atrium) and arterial blood vessels (aorta not vena cava), pancreas. Detectable levels of expression were also observed in the skeletal muscle, lung, thyroid and adrenal glands, kidney, upper portions of the gastrointestinal tract (oesophagus, stomach and small intestine but not colonic tissue) and spinal cord (but not in the cortical or cerebellar samples isolated). No detectable transcripts were derived from hepatic, bladder, adipose tissue or splenic tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (lower panel) by Southern analysis using full-length UT receptor cDNA probe.
    Mouse Brain Cdna Template, supplied by TaKaRa, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa double stranded cdna templates
    Induction of Balu-4 , Brsa-25 , Brsa-43 , Brsa-47 , Brsa-109 , and Brsa-118 mRNA by 1,000 ppb of Mn 2+ . Twenty micrograms of total RNA used in <t>SSH</t> was subjected to denaturing gel electrophoresis and hybridized with radioactively labeled probes prepared from <t>cDNA</t> clone fragments. Autoradiographs of the RNA blotting are shown on the left. Relative RNA levels are plotted on the right. The percentage of the relative amount of mRNA estimated by gel blot intensities was calculated based on relative levels of 18S rRNA.
    Double Stranded Cdna Templates, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    TaKaRa cdna template
    Knocking down β-laminin exacerbates ITGA7-mediated apoptosis. A: Semiquantitative <t>RT-PCR</t> analysis of β2-laminin in PIF1, PIF3, DIF1, and DIF5 cells. These cells were treated with tetracycline and siRNA (125 pmol/10 6 cells) specific for β2-laminin (siLam) or scrambled controls (scr). The <t>cDNA</t> templates from 1 μg total RNA of these cells were then diluted as indicated, and amplified in a PCR reaction by using primers specific for β2-laminin and β-actin. B: Representative FACS analysis of cell death of replicate samples from A, stained with fluorescein-conjugated Annexin V and propidium iodide, and analyzed in a LSC-II flow cytometer.
    Cdna Template, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 298 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cdna template/product/TaKaRa
    Average 94 stars, based on 298 article reviews
    Price from $9.99 to $1999.99
    cdna template - by Bioz Stars, 2020-09
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    Image Search Results


    RNA isolation, cDNA synthesis, and real-time PCR quantification

    Journal:

    Article Title: Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression, and regulation by cannabinoid receptor ligands

    doi: 10.1111/j.1601-183X.2009.00498.x

    Figure Lengend Snippet: RNA isolation, cDNA synthesis, and real-time PCR quantification

    Article Snippet: The tissue cDNA templates were synthesized from human RNA preparations that consist of intact RNA with no genomic DNA contamination (Clontech, Mountain View, CA).

    Techniques: Isolation, Real-time Polymerase Chain Reaction

    Tissue distribution of the mouse and monkey UT receptor: (a) Tissue distribution of mouse UT receptor cDNA transcripts by RT – PCR revealed expression within cardiac and vascular (thoracic but not abdominal aorta) tissue in addition to bladder and pancreas. Trace levels of expression are also observed in skeletal muscle, oesophagus, lung and adipose tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (Lower panel) by Southern analysis using full-length UT receptor cDNA probe. (b) Tissue distributions of monkey UT receptor cDNA transcripts by RT – PCR revealed expression within heart (ventricle > atrium) and arterial blood vessels (aorta not vena cava), pancreas. Detectable levels of expression were also observed in the skeletal muscle, lung, thyroid and adrenal glands, kidney, upper portions of the gastrointestinal tract (oesophagus, stomach and small intestine but not colonic tissue) and spinal cord (but not in the cortical or cerebellar samples isolated). No detectable transcripts were derived from hepatic, bladder, adipose tissue or splenic tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (lower panel) by Southern analysis using full-length UT receptor cDNA probe.

    Journal: British Journal of Pharmacology

    Article Title: Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey

    doi: 10.1038/sj.bjp.0704671

    Figure Lengend Snippet: Tissue distribution of the mouse and monkey UT receptor: (a) Tissue distribution of mouse UT receptor cDNA transcripts by RT – PCR revealed expression within cardiac and vascular (thoracic but not abdominal aorta) tissue in addition to bladder and pancreas. Trace levels of expression are also observed in skeletal muscle, oesophagus, lung and adipose tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (Lower panel) by Southern analysis using full-length UT receptor cDNA probe. (b) Tissue distributions of monkey UT receptor cDNA transcripts by RT – PCR revealed expression within heart (ventricle > atrium) and arterial blood vessels (aorta not vena cava), pancreas. Detectable levels of expression were also observed in the skeletal muscle, lung, thyroid and adrenal glands, kidney, upper portions of the gastrointestinal tract (oesophagus, stomach and small intestine but not colonic tissue) and spinal cord (but not in the cortical or cerebellar samples isolated). No detectable transcripts were derived from hepatic, bladder, adipose tissue or splenic tissue. (Middle panel) Amplification of GAPDH cDNA did not differ significantly between tissues. The specificity of the RT – PCR amplification of UT receptor transcripts was confirmed (lower panel) by Southern analysis using full-length UT receptor cDNA probe.

    Article Snippet: These primers were used to obtain the full-length preproU-II complementary DNA (cDNA) clone from mouse brain cDNA template (Clonetech) by polymerase chain reaction (PCR).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Isolation, Derivative Assay

    Tissue distribution of the mouse and monkey U-II. (a): Tissue distribution of mouse preproU-II cDNA transcripts by RT – PCR revealed expression within heart, thoracic aorta, testes, brain, skeletal muscle, liver, kidney and spleen (upper panel). Negligible expression of preproU-II was observed in the mouse gastrointestinal tract (stomach, oesophagus, small intestine and colon), bladder, pancreas, adrenal, lung and adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel). (b) Tissue distribution of monkey preproU-II cDNA transcripts by RT – PCR revealed expression within heart (ventricle and atrium), thoracic aorta, CNS (spinal cord, cerebellum and cortex), skeletal muscle, kidney, liver and spleen (upper panel). No detectable transcripts were derived from vena cava, endocrine tissues including thyroid, pancreas and adrenal glands, lung, gastrointestinal tissue (oesophagus, stomach, small intestine, colon), bladder or adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel).

    Journal: British Journal of Pharmacology

    Article Title: Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey

    doi: 10.1038/sj.bjp.0704671

    Figure Lengend Snippet: Tissue distribution of the mouse and monkey U-II. (a): Tissue distribution of mouse preproU-II cDNA transcripts by RT – PCR revealed expression within heart, thoracic aorta, testes, brain, skeletal muscle, liver, kidney and spleen (upper panel). Negligible expression of preproU-II was observed in the mouse gastrointestinal tract (stomach, oesophagus, small intestine and colon), bladder, pancreas, adrenal, lung and adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel). (b) Tissue distribution of monkey preproU-II cDNA transcripts by RT – PCR revealed expression within heart (ventricle and atrium), thoracic aorta, CNS (spinal cord, cerebellum and cortex), skeletal muscle, kidney, liver and spleen (upper panel). No detectable transcripts were derived from vena cava, endocrine tissues including thyroid, pancreas and adrenal glands, lung, gastrointestinal tissue (oesophagus, stomach, small intestine, colon), bladder or adipose tissue. Amplification of GAPDH cDNA did not differ significantly between tissues (middle panel). The specificity of the RT – PCR amplification of preproU-II transcripts was confirmed by Southern analysis using full-length preproU-II cDNA probe (lower panel).

    Article Snippet: These primers were used to obtain the full-length preproU-II complementary DNA (cDNA) clone from mouse brain cDNA template (Clonetech) by polymerase chain reaction (PCR).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Derivative Assay

    Induction of Balu-4 , Brsa-25 , Brsa-43 , Brsa-47 , Brsa-109 , and Brsa-118 mRNA by 1,000 ppb of Mn 2+ . Twenty micrograms of total RNA used in SSH was subjected to denaturing gel electrophoresis and hybridized with radioactively labeled probes prepared from cDNA clone fragments. Autoradiographs of the RNA blotting are shown on the left. Relative RNA levels are plotted on the right. The percentage of the relative amount of mRNA estimated by gel blot intensities was calculated based on relative levels of 18S rRNA.

    Journal: Applied and Environmental Microbiology

    Article Title: Identification of Genes Associated with Morphology in Aspergillus niger by Using Suppression Subtractive Hybridization

    doi: 10.1128/AEM.70.4.2474-2485.2004

    Figure Lengend Snippet: Induction of Balu-4 , Brsa-25 , Brsa-43 , Brsa-47 , Brsa-109 , and Brsa-118 mRNA by 1,000 ppb of Mn 2+ . Twenty micrograms of total RNA used in SSH was subjected to denaturing gel electrophoresis and hybridized with radioactively labeled probes prepared from cDNA clone fragments. Autoradiographs of the RNA blotting are shown on the left. Relative RNA levels are plotted on the right. The percentage of the relative amount of mRNA estimated by gel blot intensities was calculated based on relative levels of 18S rRNA.

    Article Snippet: Poly(A)+ RNA (1 μg) pooled from different treatments described in the “SSH” section was used to synthesize anchored, double-stranded cDNA templates for amplification of the 5′ and 3′ ends of cDNA clones via a Marathon cDNA amplification kit (Clontech).

    Techniques: Nucleic Acid Electrophoresis, Labeling, Western Blot

    Effect of antisense expression of Brsa-25 on A. niger morphology formation. (A) Diagram of the plasmid pZD570 containing the Brsa-25 gene in antisense orientation. The pGpdA corresponds to the promoter of the glyceraldehyde-3-phosphate dehydrogenase of A. nidulans. TtrpC is the A. nidulans TrpC transcription terminator. This plasmid also contains the hph gene of E. coli , which confers hygromycin resistance. (B) Microscopic observation of the morphology of the transgenic control (TAN-2811) containing the transgene expression vector with only the promoter ( pGpdA ) and terminator ( TtrpC ) and the selected Brsa-25 antisense transgenic line (antisense Brsa-25-3) after 60-h culture at 30°C and 250 rpm. (C) The RNA gel blot analysis of steady-state mRNA levels of Brsa-25 in antisense suppression strains. Total RNA was isolated from 60-h cultures of wild-type A. niger (lane 1), transgenic control strains (lane 2), antisense strain Brsa-25-3, -5, and -8 (lanes 3 to 5). Twenty micrograms of total RNA was loaded on each lane and hybridized with the radioactive labeled probe of the Brsa-25 SSH cDNA fragment. The same blot was stripped and hybridized with 18S rRNA for equivalent loading.

    Journal: Applied and Environmental Microbiology

    Article Title: Identification of Genes Associated with Morphology in Aspergillus niger by Using Suppression Subtractive Hybridization

    doi: 10.1128/AEM.70.4.2474-2485.2004

    Figure Lengend Snippet: Effect of antisense expression of Brsa-25 on A. niger morphology formation. (A) Diagram of the plasmid pZD570 containing the Brsa-25 gene in antisense orientation. The pGpdA corresponds to the promoter of the glyceraldehyde-3-phosphate dehydrogenase of A. nidulans. TtrpC is the A. nidulans TrpC transcription terminator. This plasmid also contains the hph gene of E. coli , which confers hygromycin resistance. (B) Microscopic observation of the morphology of the transgenic control (TAN-2811) containing the transgene expression vector with only the promoter ( pGpdA ) and terminator ( TtrpC ) and the selected Brsa-25 antisense transgenic line (antisense Brsa-25-3) after 60-h culture at 30°C and 250 rpm. (C) The RNA gel blot analysis of steady-state mRNA levels of Brsa-25 in antisense suppression strains. Total RNA was isolated from 60-h cultures of wild-type A. niger (lane 1), transgenic control strains (lane 2), antisense strain Brsa-25-3, -5, and -8 (lanes 3 to 5). Twenty micrograms of total RNA was loaded on each lane and hybridized with the radioactive labeled probe of the Brsa-25 SSH cDNA fragment. The same blot was stripped and hybridized with 18S rRNA for equivalent loading.

    Article Snippet: Poly(A)+ RNA (1 μg) pooled from different treatments described in the “SSH” section was used to synthesize anchored, double-stranded cDNA templates for amplification of the 5′ and 3′ ends of cDNA clones via a Marathon cDNA amplification kit (Clontech).

    Techniques: Expressing, Plasmid Preparation, Transgenic Assay, Western Blot, Isolation, Labeling

    Knocking down β-laminin exacerbates ITGA7-mediated apoptosis. A: Semiquantitative RT-PCR analysis of β2-laminin in PIF1, PIF3, DIF1, and DIF5 cells. These cells were treated with tetracycline and siRNA (125 pmol/10 6 cells) specific for β2-laminin (siLam) or scrambled controls (scr). The cDNA templates from 1 μg total RNA of these cells were then diluted as indicated, and amplified in a PCR reaction by using primers specific for β2-laminin and β-actin. B: Representative FACS analysis of cell death of replicate samples from A, stained with fluorescein-conjugated Annexin V and propidium iodide, and analyzed in a LSC-II flow cytometer.

    Journal: The American Journal of Pathology

    Article Title: Integrin Alpha 7 Interacts with High Temperature Requirement A2 (HtrA2) to Induce Prostate Cancer Cell Death

    doi: 10.2353/ajpath.2010.091026

    Figure Lengend Snippet: Knocking down β-laminin exacerbates ITGA7-mediated apoptosis. A: Semiquantitative RT-PCR analysis of β2-laminin in PIF1, PIF3, DIF1, and DIF5 cells. These cells were treated with tetracycline and siRNA (125 pmol/10 6 cells) specific for β2-laminin (siLam) or scrambled controls (scr). The cDNA templates from 1 μg total RNA of these cells were then diluted as indicated, and amplified in a PCR reaction by using primers specific for β2-laminin and β-actin. B: Representative FACS analysis of cell death of replicate samples from A, stained with fluorescein-conjugated Annexin V and propidium iodide, and analyzed in a LSC-II flow cytometer.

    Article Snippet: PCR was performed on the cDNA template from the donor prostate (Clontech) by using the following conditions: 94°C for 1 minute followed by 35 cycles of 94°C for 30 seconds, 68°C for 3 minutes, and a final 3-minute extension step at 68°C.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, FACS, Staining, Flow Cytometry, Cytometry