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| 1. |
Iwuchukwu, O.F., Ajetunmobi, J., Ung, D. and Nagar, S.
(2009)
Characterizing the effects of common UDP glucuronosyltransferase (UGT) 1A6 and UGT1A1 polymorphisms on cis- and trans-resveratrol glucuronidation.
Drug Metab. Dispos.
37
,
1726–1732
.
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| |
Notes:
This study examined the genotype-phenotype correlation of the two major UGT isoforms, UGT1A1 and UGT1A6, involved in resveratrol metabolism. Genomic DNA was isolated from 30mg human liver tissue samples (normal and metastatic) using the Wizard® SV Genomic DNA Purification System. The purified DNA was eluted with 65°C water and 200–400ng of eluted DNA was used in a PCR-RFLP UGT1A6 genotyping assay. Amplification was carried out using PCR Master Mix in a final volume of 50µl, and the amplimers digested with appropriate restriction enzymes.
(0004018) |
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Products: PCR Master Mix (2X) | Wizard® SV Genomic DNA Purification System |
| 2. |
Gill, M.B., Wright, D.E., Smith, C.M., May, J.S. and Stevenson, P.G.
(2009)
Murid herpesvirus-4 lacking thymidine kinase reveals route-dependent requirements for host colonization.
J. Gen. Virol.
90
,
1461–1470
.
|
| |
Notes:
The authors examined the role of thymidine kinase (TK) in establishing a herpesvirus infection via the upper respiratory tract. DNA was purified from ex vivo organs of female BALB/c mice infected with a murid herpesvirus-4 (MuHV-4) TK knockout using the Wizard® Genomic DNA Purification Kit. Real-time PCR was used with 50–80ng of purified DNA to determine viral load of the animals.
(0004015) |
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Products: Wizard® Genomic DNA Purification Kit | Wizard® Genomic DNA Purification Kit |
| 3. |
Acke, E., McGill, K., Golden, O., Jones, B.R., Fanning, S. and Whyte, P.
(2009)
Prevalence of thermophilic Campylobacter species in household cats and dogs in Ireland.
Vet. Rec.
164
,
44–47
.
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| |
Notes:
To examine the prevalence of Campylobacter species in asymtomatic carriers that can pass the bacteria onto humans, rectal swabs were collected from 147 dogs and 35 cats in Ireland and cultured on various diagnostic plates. The Wizard® Genomic DNA Purification System was used to isolate DNA from any suspect Campylobacter cultures. The purified DNA was used in multiplex PCR and RFLP to determine which species of Campylobacter was present.
(0004016) |
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Products: Wizard® Genomic DNA Purification Kit | Wizard® Genomic DNA Purification Kit |
| 4. |
Turci, M., Sardaro, M.L.S., Visioli, G., Maestri, E., Marmiroli, M. and Marmiroli, N.
(2009)
Evaluation of DNA extraction procedures for traceability of various tomato products.
Food Control
[epub ahead of print]
,
.
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| |
Notes:
In this study, the authors wanted to examine the ability to trace the origin of tomato goods from fresh to processed. They tested several DNA extraction procedures for fresh tomato, tomato sauce, tomato puree, tomato pulp, whole peeled S. Marzano PDO (Protected Designation of Origin) tomato, whole peeled tomato, tomato concentrate and ‘‘Arrabbiata sauce”. Homogenized material (200mg) was extracted in three replicates using seven different methods including the Wizard® DNA Clean-Up System. The DNA extracted was then analyzed by agarose gel electrophoresis, quantified and tested in PCR using SSR loci. The authors concluded that the Wizard® DNA Clean-Up System was the most effective of the DNA extraction methods tested and yielded the greatest number of successful amplification reactions with lowest investment of personnel time and money.
(0004003) |
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Products: Wizard® DNA Clean-Up Resin | Wizard® DNA Clean-Up System |
| 5. |
Yu, M.J., Miller, R.L., Uawithya, P., Rinschen, M.M., Khositseth, S., Braucht, D.W., Chou, C.L., Pisitkun, T., Nelson, R.D. and Knepper, M.A.
(2009)
Systems-level analysis of cell-specific AQP2 gene expression in renal collecting duct.
Proc. Natl. Acad. Sci. U S A
106
,
2441–2446
.
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| |
Notes:
The authors used a systems biology approach to examine the transcriptional regulation of water channel aquaporin-2 (AQP2). A 1,511bp fragment from the 5´-flanking region of the mouse AQP2 gene was amplified from mouse tail DNA and cloned into the pGEM®-T Vector. This construct was then digested with two restriction enzymes and cloned into a double-digested pGL3-Basic Vector. Full length Elf3, Elf5 and Ehf cDNA, members of the ETS family of transcriptional regulators, were amplified, sequenced and ligated into the pTARGET™ Mammalian Expression Vector. LLCPK1 cells were cotransfected with AQP2-pGL3 reporter and one of the pTARGET™ constructs. Reporter activity was measured using 20µl of cell lysate in a luciferase assay.
(0004033) |
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Products: pGEM®-T Vector | pGEM®-T Vector System I | pGEM®-T Vector System II | pGL3 Basic Vector | pTARGET™ Mammalian Expression Vector System | pTARGET™ Vector |
| 6. |
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
.
|
| |
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 |
| 7. |
Terui, Y., Mishima, Y., Sugimura, N., Kojima, K., Sakurai, T., Mishima, Y., Kuniyoshi, R., Taniyama, A., Yokoyama, M., Sakajiri, S., Takeuchi, K., Watanabe, C., Takahashi, S., Ito, Y. and Hatake, K.
(2009)
Identification of CD20 C-terminal deletion mutations associated with loss of CD20 expression in non-Hodgkin's lymphoma.
Clin. Cancer Res.
15
,
2523–2530
.
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| |
Notes:
The researchers examined if a CD20 mutation would affect resistance to rituximab, an adjuct cancer therapy drug used for CD20-positive B-cell lymphoma. CD20 PCR products amplified from genomic DNA were cloned into the pTARGET™ Mammalian Expression Vector. These CD20 mutant constructs were stably introduced into K562 chronic myelogenous leukemia cells by electroporation and selected using G-418. One microgram of CD20 mutant construct DNA was transcribed and translated using an in vitro translation kit from Promega.
(0004032) |
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Products: pTARGET™ Mammalian Expression Vector System | pTARGET™ Vector |
| 8. |
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 |
| 9. |
Rahmani, F., Hummel, M., Schuurmans, J., Wiese-Klinkenberg, A., Smeekens, S. and Hanson, J.
(2009)
Sucrose control of translation mediated by an upstream open reading frame-encoded peptide.
Plant Physiol.
150
,
1356–1367
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Notes:
The authors were wanted to study the upstream open reading frame 2 (uORF2) of the 5’ leader of bZIP11 mRNA, which has a role in sucrose regulation. The whole 5’ leader fragment of bZIP11 was subcloned into the pALTER® Vector and amino acid substitutions were introduced using the Altered Sites® II in vitro Mutagenesis System. The pGEM®-T Easy Vector was used to clone two PCR fragments that were then subcloned using restriction enzymes to create a fusion of uORF2 to a different 5’ leader. Arabidopsis seedlings were transformed via particle bombardment. 20mg of plant tissue was ground in Passive Lysis Buffer, centrifuged, and 20µl of the supernatant was assessed for reporter gene expression using the Dual-Luciferase® Reporter Assay System.
(0004023) |
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Products: Altered Sites® II in vitro Mutagenesis System | Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pALTER®-1 Vector | pGEM®-T Vector | pGEM®-T Vector System I | pGEM®-T Vector System II |
| 10. |
Sampathi, S., Bhusari, A., Shen, B. and Chai, W.
(2009)
Human flap endonuclease I is in complex with telomerase and is required for telomerase-mediated telomere maintenance.
J. Biol. Chem.
284
,
3682–3690
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| |
Notes:
The authors explored the role of a DNA replication factor, flap endonuclease I (FEN1), in regulating telomerase activity in mammalian cells. PCR was used to add a myc tag to the N terminus of FEN1 cDNA. The amplimer was gel purified, digested with NheI and SmaI, and cloned into the same sites in the pCI-neo Mammalian Expression Vector. The insert was confirmed by sequencing.
(0004030) |
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Products: pCI-neo Mammalian Expression Vector |
| 11. |
Wang, P.Y., Neretti, N., Whitaker, R., Hosier, S., Chang, C., Lu, D., Rogina, B., and Helfand, S.L.
(2009)
Long-lived Indy and calorie restriction interact to extend life span.
Proc. Natl. Acad. Sci. U S A
106(23)
,
9262-7
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| |
Notes:
These authors investigated the relationship between calorie restriction and INDY expression on lifespan in Drosophila melanogaster. They showed that calorie restriction downregulates INDY expression in normal flies. INDY mutants on a normal diet had increased lifespan that was not extended further by calorie restriction. The INDY long-lived flies also shared several phenotypic characteristics with normal flies on a calorie-restricted diet. The authors then demonstrated that the phenotypic effects of the INDY mutation were not caused by reduced ability to intake food. The results show that INDY and calorie restriction interact to extend lifespan, and that decreased INDY expression induces a calorie-restriction-like status. During the study, the Maxwell 16 Tissue DNA Purification System was used to isolate DNA from Drosophila. The DNA was used in PCR analyses to confirm the absence of Wolbachia DNA in the experimental lines.
(0004001) |
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Products: Maxwell® 16 Instrument | Maxwell® 16 Tissue LEV Total RNA Purification Kit |
| 12. |
Gaitanis, G., Velegraki, A., Alexopoulos, E.C., Kapsanaki-Gotsi, E., Zisova, L., Ran, Y., Zhang, H., Arsenis, G., Bassukas, I.D. and Faergemann, J.
(2009)
Malassezia furfur fingerprints as possible markers for human phylogeography.
ISME J.
3
,
498–502
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| |
Notes:
The authors examined Malassezia furfur strains isolated from Scandinavians permanently residing in Greece and clinical isolates from Greece, Bulgaria and China to determine if there was an association between strain and the host’s geographical origin and any underlying skin conditions. Prior to strain identification by PCR fingerprinting, M. furfur DNA was isolated using the Maxwell® 16 Instrument and a modified Maxwell® 16 Cell DNA Purification protocol. A single 2–3mm diameter yeast colony yielded 20ng of DNA.
(0003959) |
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Products: Maxwell® 16 Cell DNA Purification Kit |
| 13. |
Mancuso, M., Pasquali, E., Leonardi, S., Tanori, M., Rebessi, S., Di Majo, V., Pazzaglia, S., Toni, M.P., Pimpinellam M., Covelli, V. and Saran, A.
(2008)
Oncogenic bystander radiation effects in Patched heterozygous mouse cerebellum.
Proc. Natl. Acad. Sci. U S A
105
,
12445-12450
.
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| |
Notes:
To examine radiation-bystander responses in neonatal mouse cerebellum, heterozygous radiosensitive Patched-1 (Ptch1) mice were exposed to either whole body (WB) x-rays or shielded head/rest of body (SH) irradiation. Genomic DNA was isolated from tumors and normal tissue using the Wizard® SV Genomic DNA Purification System. Loss of heterozygosity was tested using PCR and sequencing of exon 23 of the Ptch1 gene.
(0003940) |
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Products: Wizard® SV Genomic DNA Purification System |
| 14. |
Carapelli, A., Comandi, S., Convey, P., Nardi, F. and Frati, F.
(2008)
The complete mitochondrial genome of the Antarctic springtail Cryptopygus antarcticus (Hexapoda: Collembola).
BMC Genomics
9
,
315
.
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Notes:
To sequence the mitochondrial genome one of the most widespread and common collembolan species of Antarctica, springtail Cryptopygus antarcticus. Specimens were collected from Killingbeck I during a 2002 polar expedition and frozen in liquid nitrogen. The Wizard® SV Genomic Purification System was used to extract total DNA from the samples and the complete mitochondrial genome was amplified twice, first with universal primers and sequenced, and then using long PCR with specific primers. The long PCR products were mechanically sheared, blunt end repaired and purified using the Wizard® SV Gel and PCR Clean-Up System. The fragments were then cloned, transformed and sequenced.
(0003976) |
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Products: Wizard® SV Gel and PCR Clean-Up Start-Up Kit, 110V Electrical (500 preps, manifold and free vacuum pump) | Wizard® SV Gel and PCR Clean-Up Start-Up Kit, 220V Electrical (500 preps, manifold and free vacuum pump) | Wizard® SV Gel and PCR Clean-Up Start-Up Kit, 230V Electrical (500 preps, manifold and free vacuum pump) | Wizard® SV Gel and PCR Clean-Up System | Wizard® SV Genomic DNA Purif. Start-Up Kit, 110V Electrical (500 preps, manifold and free vacuum pump) | Wizard® SV Genomic DNA Purif. Start-Up Kit, 220V Electrical (500 preps, manifold and free vacuum pump) | Wizard® SV Genomic DNA Purif. Start-Up Kit, 230V Electrical (500 preps, manifold and free vacuum pump) | Wizard® SV Genomic DNA Purification System |
| 15. |
Nagase, T., Yamakawa, H., Tadokoro, S., Nakajima, D., Inoue, S., Yamaguchi, K., Itokawa, Y., Kikuno, R.F., Koga, H. and Ohara, O.
(2008)
Exploration of human ORFeome: High-throughput preparation of ORF clones and efficient characterization of their protein products.
DNA Research
15
,
137-149
.
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Notes:
These authors used the Flexi® Vector System to prepare ORF clones encoding 1929 human genes and to transfer a subset of these clones to various expression vectors for further analysis. They created HaloTag® fusion proteins and examined expression of these proteins in vitro and in COS7 and HEK293 cells. They also performed comparisons between the Flexi® System and Gateway® cloning system, specifically examining the effects of flanking sequences on protein expression in in vitro translation systems and confirming that the cellular localization of the HaloTag® fusion proteins was consistent with results obtained using GFP-fusions.
(0003800) |
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Products: Anti-HaloTag® pAb | Dual-Glo® Luciferase Assay System | Flexi® System, Entry/Transfer | Flexi® System, Transfer | GloMax® 96 Microplate Luminometer w/Dual Injectors | Monster Green® Fluorescent Protein phMGFP Vector | pF1K T7 Flexi® Vector | pGL4.10[luc2] Vector | TNT® SP6 High-Yield Wheat Germ Protein Expression System | T7 RiboMAX™ Express Large Scale RNA Production System | Wheat Germ Extract Plus | Wizard® SV 96 PCR Clean-Up System |
| 16. |
Yamada, M., Yoshida, J., Hatou, S., Yoshida, T. and Minagawa, Y.
(2008)
Mutations in the quinolone resistance determining region in Staphylococcus epidermidis recovered from conjunctiva.
Br. J. Ophthalmol.
92
,
848–851
.
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Notes:
To study how mutations in the quinolone resistance determining region (QRDR) of Staphylococcus epidermidis may have a role in fluoroquinolone resistance, 138 samples of S. epidermidis were swabbed from the conjunctival sacs of 129 patients. These samples were cultured overnight in tryptic soy broth, and genomic DNA isolated using the Wizard® SV 96 Genomic DNA Purification System. One microliter of the isolated DNA was used in PCR for the QRDR genes (gyrA, gyrB, parC and parE).
(0003939) |
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Products: Wizard® SV 96 Genomic DNA Purification System |
| 17. |
El Sheikh, A.F., Poret-Peterson, A.T. and Klotz, M.G.
(2008)
Characterization of two new genes, amoR and amoD, in the amo operon of the marine ammonia oxidizer Nitrosococcus oceani ATCC 19707.
Appl. Environ. Microbiol.
74
,
312-318
.
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Notes:
These authors investigated the amo operon of the marine ammonia oxidizer Nitrosococcus oceani. The bacteria were grown at 30°C for 3 weeks in 200-400ml batch cultures in artificial seawater in the dark without shaking. Genomic DNA was isolated from cells in stationary phase using the Wizard® Genomic DNA Purification Kit. The isolated DNA was then used for PCR analysis.
(0003740) |
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Products: Wizard® Genomic DNA Purification Kit | Wizard® Genomic DNA Purification Kit |
| 18. |
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 |
| 19. |
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 |
| 20. |
Baker, S., Holt, K., van de Vosse, E., Roumagnac, P., Whitehead, S., King, E., Ewels, P., Keniry, A., Weill, F.X., Lightfoot, D., van Dissel, J.T., Sanderson, K.E., Farrar, J., Achtman, M., Deloukas, P. and Dougan, G.
(2008)
High-throughput genotyping of Salmonella enterica serovar Typhi allowing geographical assignment of haplotypes and and pathotypes within an urban District of Jakarta, Indonesia.
J. Clin. Microbiol.
46
,
1741–1746
.
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Notes:
The authors examined strains of Salmonella enterica serovar Typhi isolated from typhoid cases originating in or around Indonesia or from travelers returning from Indonesia to examine if serovar Typhi from this area has a greater level of genetic diversity compared to other countries. Genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit, diluted to 4 ng/µl and used in locus-specific PCR genotyping.
(0003980) |
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Products: Wizard® Genomic DNA Purification Kit | Wizard® Genomic DNA Purification Kit |
| 21. |
Nguyen, A.V., Thomas-Hall, S.R., Malnoë, A., Timmins, M., Mussgnug, J.H., Rupprecht, J., Kruse, O., Hankamer, B. and Schenk, P.M.
(2008)
Transcriptome for photobiological hydrogen production induced by sulfur deprivation in the green alga Chlamydomonas reinhardtii.
Eukaryot. Cell.
7
,
1965–1979
.
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Notes:
The authors analyzed the transcriptional activity of wild-type Chlamydomonas reinhardtii cultures sampled at different time points during the aerobic and anaerobic phase of the photobiological hydrogen production process under sulfur-depleted conditions. C. reinhardtii were grown in photobioreactors, carefully extracted, centrifuged and flash-frozen in liquid nitrogen. RNA was purified using the SV Total RNA Isolation System following the plant centrifugation protocol without sample grinding. The eluted RNA was quantitated and integrity checked by gel electrophoresis and qRT-PCR. Total RNA was used to synthesize labeled cDNA using the ChipShot™ Indirect Labeling and Clean-Up System. The labeled cDNA was used for probing microarrays.
(0004022) |
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Products: ChipShot™ Indirect Labeling and Clean-Up System | SV Total RNA Isolation System |
| 22. |
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 |
| 23. |
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 |
| 24. |
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 |
| 25. |
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 |