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| 1. |
Izukawa, T., Nakajima, M., Fujiwara, R., Yamanaka, H., Fukami, T., Takamiya, M., Aoki, Y., Ikushiro, S., Sakaki, T. and Yokoi, T.
(2009)
Quantitative analysis of UDP-glucuronosyltransferase (UGT) 1A and UGT2B expression levels in human livers.
Drug Metab. Dispos.
37
,
1759–1768
.
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Notes:
This study examined the expression levels of each UGT isoform in human liver and evaluated the variability between individuals. Total RNA from appropriate human tissues or various cell lines was used for RT-PCR of various human UDP-glucuronosyltransferases (UGT) cDNAs. The amplimers were cloned into the pTARGET™ Mammalian Expression Vector and verified by sequencing. The UGT vectors were linearized by restriction enzyme digestion and used for standards in real-time RT-PCR analysis.
(0004034) |
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Products: pTARGET™ Mammalian Expression Vector System | pTARGET™ Vector |
| 2. |
Wilson, P.M., Fazzone, W., LaBonte, M.J., Lenz, H.J. and Ladner, R.D.
(2009)
Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage.
Nucleic Acids Res.
37
,
78–95
.
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Notes:
The authors examined the role of p53 in modulating dUTPase promoter activity. Base substitution mutations of Sp1- and E2F-binding sites in the dUTPase promoter were performed using the GeneEditor™ in vitro Site-Directed Mutagenesis System. Each mutant was confirmed by DNA sequencing. To determine growth inhibition, HCT116 human colon cancer cells were seeded in 96-well plates at 3 × 103 cells/well and treated with 5-fluorouracil (5-FU), fluorodeoxyuridine (FUdR), oxaliplatin or in combination. After 72 hours, the CellTiter® 96 AQueous One Solution was dispensed into each well and absorbance measured. RNA was isolated from HCT116 p53+/+ and HCT116 p53-/- cells. cDNA was reverse transcribed from 200ng total RNA followed by multiplex qPCR using the Plexor™ qPCR System to amplify dUTPase, thymidylate synthase and GAPDH, a housekeeping gene. The 1.2 kb region of the dUTPase promoter upstream of the transcriptional start site was amplified by PCR and the fragment cloned into the pGL3-Basic Vector. Truncated promoters were also generated by PCR and cloned into the same vector. Drosophila SL-2 cells and HCT116 cell lines were seeded in a 24-well plate and transfected with dUTPase pGL3 promoter constructs or with pCI-Neo:p53WT, pCI-Neo:p53MUT and the empty pCI-neo Mammalian Expression Vector; all transfections included the pRL-TK Vector at a ratio of 1:10. After six hours, the cells were incubated in either fresh medium or medium containing a cytotoxic agent at the appropriate concentration. Thirty hours later, the cells were lysed, quantitated by Western blotting and 20µl of lysate analyzed with the Dual-Luciferase® Reporter Assay System. Electrophoretic mobility shift analyses (EMSA) were performed using –64 to –91 of the dUTPase-nuclear isoform transcriptional start site in the Gel Shift Assay System.
(0004031) |
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Products: CellTiter 96® AQueous One Solution Cell Proliferation Assay | CellTiter 96® AQueous One Solution Reagent | CellTiter 96® AQueous One Solution Reagent | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | Gel Shift Assay Core System | Gel Shift Assay System | GeneEditor™ in vitro Site-Directed Mutagenesis System | pCI Mammalian Expression Vector | pGL3 Basic Vector | Plexor® qPCR System | pRL-TK Vector |
| 3. |
Fekete, E., Seiboth, B., Kubicek, C.P., Szentirmai, A., Karaffa, L.
(2008)
Lack of aldose 1-epimerase in Hypocrea jecorina (anamorph Trichoderma reesei): A key to cellulase gene expression on lactose
Proc. Natl. Acad. Sci. U S A
105
,
7141-7146
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Notes:
To amplify yeast mutarotase, S. cerevisiae was used, and E. coli strain JM109 (Promega Cat.# L2001) was used for plasmid propagation. Fungal mycelia were harvested by filtration, washed, frozen and ground under liquid nitrogen. Genomic DNA was extracted using the Wizard Genomic DNA Purification System (Promega Cat.# A1120). RNA for hybridization and RT-PCR was extracted from mycelia using the SV Total RNA Isolation System (Promega Cat.# Z3101) and plasmid DNA isolated using the PureYield(TM) Plasmid Midiprep System (Cat.# A2492).
(0003919) |
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Products: JM109 Competent Cells, >107cfu/μg | PureYield™ Plasmid Midiprep System | Wizard® Genomic DNA Purification Kit | Wizard® Genomic DNA Purification Kit |
| 4. |
Flahaut, S., Vinogradov, E., Kelley, K.A., Brennan, S., Hiramatsu, K. and Lee, J.C.
(2008)
Structural and biological characterization of a capsular polysaccharide produced by Staphylococcus haemolyticus.
J. Bacteriol.
190
,
1649–1657
.
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Notes:
The authors wanted to purify and characterize the capsular polysaccharide (CP) produced by Staphylococcus haemolyticus strain JCSC1435. S. haemolyticus strains grown in TSB cultures were harvested, lysed with lysozyme and lysostaphin and genomic DNA isolated using the Wizard® Genomic DNA Purification Kit. The DNA was then used in CP gene PCR. Total RNA was isolated from exponential, postexponential, and stationary phases of S. haemolyticus growth and used in RT-PCR using the Access RT-PCR System.
(0003977) |
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Products: Access RT-PCR Introductory System | Access RT-PCR System | Wizard® Genomic DNA Purification Kit | Wizard® Genomic DNA Purification Kit |
| 5. |
Vergnano, A.M., Ferrini, F., Salio, C., Lossi, L., Baratta, M. and Merighi, A.
(2008)
The gastrointestinal hormone ghrelin modulates inhibitory neurotransmission in deep laminae of mouse spinal cord dorsal horn.
Endocrinology
149
,
2306–12
.
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Notes:
The authors investigated the expression pattern of type 1a growth hormone secretagogue receptor (type 1a GHSR), a receptor for ghrelin. In situ RT-PCR was performed on paraformaldehyde-fixed, paraffin-embedded mouse spinal cord tissue. Prior to in situ RT-PCR, tissue sections were treated with proteinase K and triethanolamine, then dewaxed. Reverse transcription was performed using the Reverse Transcription System and oligo (dT)15 primers; followed by amplification using the PCR Master Mix in the presence of 11-digoxygenin-dUTP (1mM). Amplification products were detected using an anti-digoxygenin, alkaline phosphatase-conjugated goat antibody and nitro blue tetrazolium/5-bromo-3-indolylphosphate-p-toluidine salt (NBT/BCIP).
(0003968) |
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Products: PCR Master Mix | Reverse Transcription System |
| 6. |
Staniszewska, I., Sariyer, I.K., Lecht, S., Brown, M.C., Walsh, E.M., Tuszynski, G.P., Safak, M., Lazarovici, P. and Marcinkiewicz, C.
(2008)
Integrin alpha9 beta1 is a receptor for nerve growth factor and other neurotrophins.
J. Cell Sci.
121
,
504–13
.
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Notes:
The authors investigated the ability of α9β1 integrin to act as a neurotrophin receptor and affect cell signaling pathways. As part of the study, RT-PCR was used to detect the presence of other neurotrophin receptors in their model cell line, SW480. Reverse transcription was performed using the Reverse Transcription System and 1µg of total RNA isolated using the SV Total RNA Isolation System. The resulting cDNA (5µg) was amplified for 35 cycles (β-actin as a control) or 40 cycles (TrkA and p75NTR). RT-PCR results were confirmed by Western blot analysis.
(0003884) |
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Products: GoTaq® Green Master Mix | Reverse Transcription System | SV Total RNA Isolation System |
| 7. |
Leseberg, C.H., Eissler, C.L., Wang, X., Johns, M.A., Duvall, M.R. and Mao, L.
(2008)
Interaction study of MADS-domain proteins in tomato.
J. Exp. Bot.
59
,
2253–65
.
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Notes:
The authors characterized the network of protein-protein interactions for 22 MADS-domain proteins in tomato using yeast two-hybrid and three-hybrid assays. To construct bait and prey proteins, total RNA from various tissues was reverse transcribed using the Reverse Transcription System, then amplified using PCR primers containing restriction enzyme sites for cloning into the bait and prey vectors.
(0003886) |
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Products: Reverse Transcription System |
| 8. |
Mie, M., Shimizu, S., Takahashi, F. and Kobatake, E.
(2008)
Selection of mRNA 5´-untranslated region sequence with high translation efficiency through ribosome display.
Biochem. Biophys. Res. Commun.
373
,
48–52
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Notes:
The authors developed an in vitro selection system that is based on ribosome display and favors identification of 5´-untranslated regions (UTRs) with high translation efficiencies. A 5´-UTR random library was created in which the 5´-UTRs were upstream of a polyhistidine-tag/Renilla luciferase-coding region. In vitro transcripts from this library were translated in vitro using the Flexi® Rabbit Reticulocyte Lysate System. The authors preferentially selected mRNAs with high translational efficiencies by shortening the translation time and capturing ternary complexes of mRNA, ribosome and nascent proteins. These complexes were captured using MagneHis™ Ni Particles. RNA was extracted from these complexes and used as a template in RT-PCR for the next round of selection. Before and after each round of selection, 9µl of RNA was translated in vitro, and 20µl of translated product was removed every 5 minutes to measure Renilla luciferase activity and monitor translation efficiency. Renilla luciferase was measured using the Renilla Luciferase Assay System. After two rounds of selection, RT-PCR products were cloned into a pUC18 vector, the sequences of the resulting plasmids were confirmed, and 0.5µg of plasmid was translated in vitro using the TNT® T7 Coupled Rabbit Reticulocyte Lysate System to further evaluate translation efficiency.
(0003963) |
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Products: Renilla Luciferase Assay System | Flexi® Rabbit Reticulocyte Lysate System | MagneHis™ Ni-Particles | TNT® T7 Coupled Reticulocyte Lysate System |
| 9. |
Dall'Osso, C., Guella, I., Duga, S., Locatelli, N., Paraboschi, E.M., Spreafico, M., Afrasiabi, A., Pechlaner, C., Peyvandi, F., Tenchini, M.L. and Asselta, R.
(2008)
Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern.
Haematologica
93
,
1505–1513
.
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Notes:
To examine the causes of Factor V (FV) deficiency, the authors examined transcript splicing and its mutated variations. Three regions of human FV (F5) were amplified from a healthy individual and the PCR products cloned into the pTargeT™ Mammalian Expression Vector. Three identified mutations from people with FV deficiency were introduced by site-directed mutatgenesis. All constructs were sequenced before transfection into HeLa cells. After 48 hours, the total RNA was purified and the splicing pattern of the wild type and mutant constructs were analyzed by RT-PCR. The mutant constructs were also transfected into HepG2 cells and tested for nonsense-mediated mRNA decay (NMD) with or without NMD inhibitors (puromycin, cycloheximide, and wortmannin) using RT-PCR.
(0003992) |
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Products: pTARGET™ Mammalian Expression Vector System |
| 10. |
Wang, L., Zheng, G.G., Ma, C.H., Lin, Y.M., Zhang, H.Y., Ma, Y.Y., Chong, J.H. and Wu, K.F.
(2008)
A special linker between macrophage and hematopoietic malignant cells: membrane form of macrophage colony-stimulating factor.
Cancer Res.
68
,
5639–5647
.
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Notes:
To examine the role of the membrane form of macrophage colony–stimulating factor(mM-CSF) in the hematopoietic system, RT-PCR was used amplify the cDNA of human mM-CSF from J6-1 cells, a human leukemia cell line. The PCR product was digested and cloned into the pTargeT™ Mammalian Expression Vector. After sequencing to verify the sequence, the construct and empty pTargeT™ Mammalian Expression Vector were purified and used to transfect Namalwa and Ramos cells, human Burkitt’s lymphoma cell lines, in 24-well plates. The transfected cells were then selected for stable expression of the transfected vector using 1.4 mg/ml G418. Expression of mM-CSF and neomycin (in the empty vector) was confirmed using RT-PCR. These cells were injected into mice and the oncogenicity of the cells determined using antibody staining of tissues.
(0003989) |
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Products: pTARGET™ Mammalian Expression Vector System |
| 11. |
Haraguchi, M., Okubo, T., Miyashita, Y., Miyamoto, Y., Hayashi, M., Crotti, T.N., McHugh, K.P. and Ozawa, M.
(2008)
Snail regulates cell-matrix adhesion by regulation of the expression of integrins and basement membrane proteins.
J. Biol. Chem.
283
,
23514–23
.
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Notes:
Snail is a transcriptional repressor of E-cadherin that enhances both cell attachment and cell detachment in Madin Darby canine kidney (MDCK) and A4231 cells. To investigate this effect, the authors used Western blot analysis and RT-PCR to monitor protein and mRNA levels of the major adhesive proteins expressed in epithelial cells: laminin, heparin sulfate proteoglycan and collagens. For RT-PCR, total RNA was isolated from transiently transfected snail-expressing MDCK and A431 cells and untransfected cells, then reverse transcribed. The resulting cDNA was amplified by PCR using GoTaq® DNA Polymerase; glyceraldehyde-3-phosphate dehydrogenase was amplified as an internal control. The ability of Snail to regulate the integrin αV promoter was also examined by cloning the promoter and several promoter deletions upstream of a firefly luciferase reporter gene in the pGL3-Basic Vector. Each of these constructs (1µg) and 20ng of pRL-CMV Vector were transfected into MDCK and MDCK/snail cells, and luminescence was measured using the Dual Luciferase Assay System.
(0003882) |
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Products: Dual-Luciferase® Reporter Assay System | GoTaq® DNA Polymerase | pGL3-Basic Vector |
| 12. |
Sal, M.S., Li, C., Motalab, M.A., Shibata, S., Aizawa, S. and Charon, N.W.
(2008)
Borrelia burgdorferi uniquely regulates its motility genes and has an intricate flagellar hook-basal body structure.
J. Bacteriol.
190
,
1912–21
.
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Notes:
The authors investigated gene transcription within periplasmic flagella of Borrelia burgdorferi, which are composed of a basal body, hook and filament, to determine if hook formation influences flagellin gene expression. They used insertion mutagenesis to construct strains with mutated versions of the hook structural gene flgE that were disrupted by a kanamycin-resistance cassette. The flgE gene and antibiotic-resistance cassette were amplified by PCR and cloned into the pGEM®-T Vector. To assess the effect of flgE disruption on the transcription of filament proteins FlaA and FlaB, quantitative RT-PCR was performed; enolase was used as an internal control. Negative controls without the reverse transcriptase were included for each sample.
(0003885) |
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Products: pGEM®-T Easy Vector System I | Reverse Transcription System |
| 13. |
Scorpio, D.G., Leutenegger, C., Berger, J., Barat, N., Madigan, J.E. and Dumler, J.S.
(2008)
Sequential analysis of Anaplasma phagocytophilum msp2 transcription in murine and equine models of human.
Clin. Vaccine Immunol.
15
,
418–424
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Notes:
The authors examined the pattern of Anaplasma phagocytophilum msp2 expression, a gene that modulates with little immune pressure and has decreased virulence with prolonged in vitro passage. C57BL/6J mice were inoculated with HL-60 cells infected with low-passage (passage 5) or high-passage (passage 26). Blood samples were taken 2–21 days post-inoculation, and total RNA was isolated. The purified RNA was subjected to RT-PCR, cloned into the pGEM®-T Easy Vector, transformed and plated. Plasmids were purified using the Wizard® SV 96 Plasmid DNA Purification System, and the insert size analyzed after EcoRI digestion. The inserts were sequenced, aligned with A. phagocytophilum Webster strain msp2 references using ClustalX and the diversity of msp2 transcripts divided into low- or high-passage bacteria.
(0003975) |
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Products: pGEM®-T Easy Vector System I | pGEM®-T Easy Vector System II | Wizard® SV 96 Plasmid DNA Purification System |
| 14. |
Wang, Y., Ladunga, I., Miller, A.R., Horken, K.M., Plucinak, T., Weeks, D.P. and Bailey, C.P.
(2008)
The small ubiquitin-like modifier (SUMO) and SUMO-conjugating system of Chlamydomonas reinhardtii.
Genetics
179
,
177-192
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Notes:
These authors used computational biology to screen the genome of the alga Chlamydomonas reinhardtii for SUMO (small ubiquitin-like modifier) homologs. They identified several SUMO and SUMO-like sequences. One of these proteins, crSUMO96, which was recognized by the A. thaliana anti-SUMO antibody, was studied in detail. During their studies, the authors used the PureYield™ RNA Midiprep System to isolate total RNA from C. reinhardtii cells. This RNA was used in real-time RT-RCR assays to detect mRNA transcripts for the various SUMO-like proteins. The Plexor® Two-Step qRT-PCR System was used for the real-time assays. For expression studies, cDNA encoding the various proteins was amplified and subcloned into the pGEM®-T Easy Vector before transfer into an expression vector.
(0003875) |
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Products: pGEM®-T Easy Vector System I | pGEM®-T Easy Vector System II | Plexor® Two-Step qRT-PCR System | PureYield™ RNA Midiprep System |
| 15. |
Zhao, Z., Peytavi, R., Diaz-Quijada, G.A., Picard, F.J., Huletsky, A., Leblanc, E., Frenette, J., Boivin, G., Veres, T., Dumoulin, M.M. and Bergeron, M.G.
(2008)
Plastic polymers for efficient DNA microarray hybridization: application to microbiological diagnostics.
J. Clin. Microbiol.
46
,
3752–3758
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Notes:
A plastic support suitable for use in microfluidic systems for highly sensitive DNA microarray hybridizations was developed and tested. Human DNA from Hsap-11 cells was isolated using the MagneSil® KF, Genomic System on a KingFisher ML instrument. Ten nanograms of the isolated DNA was used in RT-PCR.
(0004021) |
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Products: MagneSil® KF, Genomic System |
| 16. |
Liu, Q., Fu, H., Sun, F., Zhang, H., Tie, Y., Zhu, J., Xing, R., Sun, Z. and Zheng, X.
(2008)
miR-16 family induces cell cycle arrest by regulating multiple cell cycle genes.
Nucleic Acids Res.
36
,
5391–404
.
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Notes:
To identify microRNA targets, the authors created a Drosha-knockdown cell line and confirmed depletion of Drosha and three randomly selected miRNAs in these cells by quantitative RT-PCR, using β-actin as a control. The reverse transcription step was performed using the ImProm-II™ Reverse Transcription System. The authors then performed microarray analysis to monitor expression of transcripts to determine which were upregulated as a result of Drosha depletion; cRNA used in these microarray experiments was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. Cyclin D1 was identified as a potential miRNA target. To screen miRNAs that regulate cyclin D1, the authors cloned the cyclin D1 3´ untranslated region downstream of the firefly luciferase gene of the pGL3-Control Vector and measured luciferase levels in transfected cells using the Dual Luciferase Reporter Assay System. Renilla luciferase in the pRL-TK Vector was used as a normalization control.
(0003894) |
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Products: Dual-Luciferase® Reporter Assay System | ImProm-II™ Reverse Transcriptase | pGL3-Control Vector | pRL-TK Vector | T7 RiboMAX™ Express Large Scale RNA Production System |
| 17. |
Jin, Z., Lahat, G., Korchin, B., Nguyen, T., Zhu, Q.S., Wang, X., Lazar, A.J., Trent, J., Pollock, R.E. and Lev D.
(2008)
Midkine enhances soft-tissue sarcoma growth: a possible novel therapeutic target.
Clin. Cancer Res.
14
,
5033–42
.
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Notes:
Increased expression of midkine (MK), a growth factor normally involved in neural development, is associated with several human malignancies. The authors used quantitative RT-PCR to examine mRNA levels for MK and MK receptors in various soft-tissue sarcoma (STS) cell lines. Reverse transcription was performed using 1µg of total RNA, and 2µl of cDNA was used in qPCR using the PCR Master Mix, primers specific to MK and either glyceraldehyde-3-phosphate or β-actin primers for normalization, and EvaGreen® dye. To examine the protumorigenic effects of MK, the authors incubated HT1080 and SW684 STS cell lines, which express low levels of MK, with human recombinant MK and measured cell proliferation using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. In addition, the authors examined cell proliferation in MK-stably transfected HT1080 cells. The plasmid used for stable transfections was created by reverse transcribing the MK-coding region using the ImProm-II™ Reverse Transcription System, then cloning the resulting cDNA into an expression vector.
(0003895) |
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Products: CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay |
| 18. |
Simonetti, M., Giniatullin, R., and Fabbretti, E.
(2008)
Mechanism mediating the enhanced transcription of P2X3 receptor gene by calcitonin gene related peptide in trigeminal sensory neurons.
J. Biol. Chem.
May 6
,
Epub (ahead of print)
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Notes:
These authors investigated the mechanism of action of the migraine mediator calcitonin gene-related peptide (CGRP), which is known to sensitize the P2X3 pain receptors to increase impulse flow to brain stem trigeminal nuclei. They showed that CAM KII inhibitors prevented CGRP-induced upregulation of P2X3 mRNA using real-time RT-PCR, and then confirmed that active CAM KII was involved in the signaling mechanism by staining with Anti-ACTIVE® CAM KII Antibodies. The immunoreactivity was upregulated by CGRP treatment of trigeminal neurons, and the distribution of the active CAM KII was localized to the membrane region. They then examined the mechanism of transcriptional activation of the P2X3 pain receptor genes and showed that the transcription factor CREB, which is known to be dependent on CAM KII for activation and nuclear localization, was involved. The authors also investigated the role of BDNF in the process, because CGRP is known to promote BDNF expression by trigeminal neurons, and because BDNF is thought to be involved in pain processing. They used the BDNF Emax Immunoassay System to measure BDNF levels in culture media after application of CGRP to neuronal cell cultures, and demonstrated that there was a fourfold increase in BDNF after CGRP exposure. They also showed that BDNF promoted CAMKII and CREB activation.
(0003873) |
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Products: Anti-ACTIVE® CaM KII pAb, Rabbit, (pT286) | BDNF Emax® ImmunoAssay System |
| 19. |
Kelleher, D.F., de Carvalho, C.E., Doty, A.V., Layton, M., Cheng, A.T., Mathies, L.D., Pilgrim, D. and Haag, E.S.
(2008)
Comparative genetics of sex determination: masculinizing mutations in Caenorhabditis briggsae.
Genetics
178
,
1415–29
.
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Notes:
The authors characterized masculinizing mutations of the female-promoting tra genes in Caenorhabditis briggsae (Cb-tra). Using RT-PCR, the authors monitored the levels of full-length Cb-tra mRNA and a novel splice variant; actin mRNA was amplified as a control. RT-PCR was carried out using the AccessQuick™ RT-PCR System and RNA from 5–10 worms per 50µl reaction.
(0003892) |
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Products: AccessQuick™ RT-PCR System |
| 20. |
Nanashima, N., Akita, M., Yamada, T., Shimizu, T., Nakano, H., Fan, Y. and Tsuchida, S.
(2008)
The hairless phenotype of the Hirosaki hairless rat is due to the deletion of an 80-kb genomic DNA containing five basic keratin genes.
J. Biol. Chem.
283
,
16868–75
.
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Notes:
The mutation responsible for the hairless phenotype was linked to a 80kb deletion of chromosome 7q36. Because many basic keratin genes are located at 7q36, the authors examined keratin gene expression in the Hirosaki hairless rat using RT-PCR. Expression of kb21, kb23 and kb26 was not detected, whereas other basic keratin genes were expressed. RT-PCR was performed using the AccessQuick™ RT-PCR System and 0.5µg of total RNA isolated from rat skin for 21–30 cycles.
(0003887) |
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Products: AccessQuick™ RT-PCR System |
| 21. |
Chang, Q,. Bhatia, D., Zhang, Y., Meighan, T., Castranova, V., Shi, X. and Chen, F.
(2008)
Incorporation of an internal ribosome entry site-dependent mechanism in arsenic-induced GADD45 alpha expression.
Cancer Res.
2007
,
6146–54
.
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Notes:
Trivalent arsenic (As3+) induces expression of growth arrest- and DNA damage-induced gene 45α (GADD45α), which interacts with intracellular signaling molecules involved in cell cycle regulation, apoptosis and immune response. To characterize GADD45α mRNA expression patterns, total RNA was isolated from untreated (growing) and As3+-treated (arrested) BEAS-2B cells, and GADD45α mRNA was amplified using the AccessQuick™ RT-PCR System. GADD45α mRNA levels were undetectable in growing cells but increased in a time-dependent manner in arrested cells. Reverse transcription was carried out at 45°C for 50 minutes, followed by 35 cycles of PCR.
(0003766) |
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Products: AccessQuick™ RT-PCR System |
| 22. |
Miao, Q., Sun, Y., Wei, T., Zhao, X., Zhao, K., Yan, L., Zhang, X., Shu, H. and Yang, F.
(2008)
Chymotrypsin B cached in rat liver lysosomes and involved in apoptotic regulation through a mitochondrial pathway.
J. Biol. Chem.
283
,
8218–28
.
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| |
Notes:
The authors characterized a novel caspase 8-like activity that cleaves Bid and activates the mitochondrial apoptotic pathway. This activity was purified from rat liver lysosomal extracts and later identified as chymotrypsin B (CtrB). CtrB was previously thought to be expressed only in the pancreas, but the authors were able to detect crtB RNA in total RNA from primary rat hepatocytes and a rat hepatoma cell line (RH-35) using RT-PCR and the Access RT-PCR System. To confirm the intralysosomal localization of Crt B, the authors transfected RH-35 cells with an expression vector encoding CrtB tagged with green fluorescent protein. Prior to transfection, synthesis of functional protein from the expression vector was confirmed by in vitro transcription and translation using the TNT® Coupled Transcription Translation System and [35S] methionine.
(0003889) |
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Products: Access RT-PCR System | TNT® T7 Coupled Reticulocyte Lysate System |
| 23. |
Lu, Y., Wang, J., Xu, Y., Koch, A.E., Cai, Z., Chen, X., Galson, D.L., Taichman, R.S. and Zhang, J.
(2008)
CXCL16 functions as a novel chemotactic factor for prostate cancer cells in vitro.
Mol. Cancer Res.
6
,
546–54
.
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Notes:
The authors sought to detect expression of the chemokine CXCL16 and its receptor, CXCR6, in prostate cancer cell lines and in benign and malignant prostate cancer tissues. The Access RT-PCR System was used to amplify and detect CXCL16 and CXCR6 mRNA in these cells and tissues. Each RT-PCR contained 1µg of total RNA, and amplifications were carried out for 35 cycles. Amplified products were detected on a 1.5% ethidium bromide-stained agarose gel.
(0003836) |
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Products: Access RT-PCR System |
| 24. |
Luce-Fedrow. A., Von Ohlen, T., Boyle, D., Ganta, R.R. and Chapes, S.K.
(2008)
Use of Drosophila S2 cells as a model for studying Ehrlichia chaffeensis infections.
Appl. Environ. Microbiol.
74
,
1886–91
.
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Notes:
The authors infected Drosophila S2 cells with Ehrlichia chaffeensis to determine if Drosophila is a model system to study E. chaffeensis pathogenesis. E. chaffeensis was also grown in canine macrophage-like DH82 cells, the most common host cell line. Infections were assessed by RT-PCR using the Access RT-PCR System, 0.5–1.0µg of RNA and primers specific to the E. chaffeensis 16S rRNA gene. Housekeeping genes for ribosomal protein 49 and canine glyceraldehyde-3-phosphate dehydrogenase were amplified as control targets for Drosophila and DH82 cells, respectively. Negative controls without reverse transcriptase were performed to be sure that DNA was absent from the RNA samples.
(0003888) |
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Products: Access RT-PCR System |
| 25. |
Panis, G., Méjean, V. and Ansaldi, M.
(2007)
Control and regulation of KplE1 prophage site-specific recombination: a new recombination module analyzed.
J. Biol. Chem.
282
,
21798–21809
.
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Notes:
The authors studied the defective prophage KplE1 in E. coli K12 to map the binding sites of proteins required for recombination. Prior to in vivo excision assays in two E. coli K12 strains, the presence of three DNA sequences required for recombination was confirmed by PCR using GoTaq® DNA Polymerase. In vitro excision assays were also performed using linear and supercoiled DNA substrates that were purified using the Wizard® PCR Clean-Up System. Finally the phage-encoded integraseS (IntS) mRNA was quantitated by real-time RT-PCR. The RNA template was purified from E. coli K12 using the PureYield™ RNA Midiprep System.
(0003722) |
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Products: GoTaq® DNA Polymerase | PureYield™ RNA Midiprep Start-Up Kit, 110V Electrical (100 preps, manifold, 4 elution devices and free vacuum pump) | PureYield™ RNA Midiprep Start-Up Kit, 220V Electrical (100 preps, manifold, 4 elution devices and free vacuum pump) | Wizard® SV Gel and PCR Clean-Up System |