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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. |
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 |
| 3. |
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 |
| 4. |
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 |
| 5. |
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 |
| 6. |
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 |
| 7. |
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 |
| 8. |
Oliveira, L.R., Ribeiro-Silva, A., Ramalho, L.N.Z., Simões, A.L. and Zucoloto, S.
(2008)
HPV infection in Brazilian oral squamous cell carcinoma patients and its correlation with clinicopathological outcomes
Mol. Med. Reports Atenas
1
,
123–129
.
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Notes:
In this study of the frequency of human papilloma virus (HPV) in patients with oral squamous cell carcinoma (OSCC), the MagneSil® Genomic, Fixed Tissue System was used to isolate DNA from formalin-fixed paraffin-embedded tissue samples from primary tumors and matched samples. Five microliters of genomic DNA was amplified using PCR Master Mix and primers for both a 110 bp fragment of human ß-globin gene and HPV genotype. After PCR, the product was analyzed on an 8% nondenaturing polyacrylamide gel and stained with AgNO3.
(0003938) |
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Products: MagneSil® Genomic, Fixed Tissue System | PCR Master Mix |
| 9. |
Covshoff, S., Majeran, W., Liu, P., Kolkman, J.M., van Wijk, K.J. and Brutnell, T.P.
(2008)
Deregulation of maize C4 photosynthetic development in a mesophyll cell-defective mutant.
Plant Phys.
146
,
1469–81
.
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Notes:
The authors identified the maize homolog of hcf136 (Zmhcf136), a gene involved in photosynthesis, and used an RNA blot to determine if ZmHcf136 transcripts accumulate preferentially in mesophyll cells. DNA probes for Zmhcf136 and several cell-specific markers were generated by PCR using GoTaq® Green Master Mix, gel purified and radiolabeled prior to use in the RNA blots. To examine differences in protein accumulation and localization in wildtype and hcf136 mutants, proteins from subcellular fractions were subjected to two-dimensional gel electrophoresis, and spots of interest were excised, digested with Sequencing Grade Modified Trypsin, then analyzed by electrospray ionization-tandem mass spectrometry.
(0003883) |
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Products: GoTaq® Green Master Mix | Sequencing Grade Modified Trypsin |
| 10. |
McManus, B.A., Coleman, D.C., Moran, G., Pinjon, E., Diogo, D., Bougnoux, M.E., Borecká-Melkusova, S., Bujdákova, H., Murphy, P., d'Enfert, C. and Sullivan, D.J.
(2008)
Multilocus sequence typing reveals that the population structure of Candida dubliniensis is significantly less divergent than that of Candida albicans.
J. Clin. Microbiol.
46
,
652–64
.
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Notes:
To determine the usefulness of multilocus sequence typing (MLST) in differentiating Candida species during epidemiological studies, the authors investigated the population structure of C. dubliniensis by amplifying the same 10 MLST loci found to be useful in differentiating isolates of C. albicans, a closely related species. PCRs were performed using 1.25 units of GoTaq® Flexi DNA Polymerase and 1ng of DNA template in a 50µl reaction.
(0003880) |
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Products: GoTaq® Flexi DNA Polymerase |
| 11. |
Bermejo-Alvarez, P., Rizos, D., Rath, D., Lonergan, P. and Gutiérrez-Adán, A.
(2008)
Can bovine in vitro-matured oocytes selectively process X- or Y-sorted sperm differentially?
Biol. Reprod.
79
,
594–7
.
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| |
Notes:
To determine whether oocytes are able to select X-bearing or Y-bearing spermatozoa, the authors performed in vitro fertilization of bovine oocytes with X-sorted semen, Y-sorted semen, a mixture of X- and Y-sorted semen, and unsorted semen. The gender of the resulting embryos was determined by amplifying two DNA targets: a Y chromosome-specific target for gender assignment and a bovine-specific satellite sequence as a control. PCRs were performed using GoTaq® Flexi DNA Polymerase (1 unit per 25µl reaction), and amplified products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining.
(0003881) |
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Products: GoTaq® Flexi DNA Polymerase |
| 12. |
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 |
| 13. |
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 |
| 14. |
Yu, Q., Collavo, A., Zheng, M.Q., Owen, M., Sattin, M. and Powles, S.B.
(2007)
Diversity of acetyl-coenzyme A carboxylase mutations in resistant Lolium populations: Evaluation using clethodim.
Plant Physiol.
145
,
547–558
.
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Notes:
The authors characterized mutations in the acetyl-coenzyme A carboxylase (ACCase) gene that confer resistance to the herbicide clethodim in the grass weed Lolium rigidum. The ACCase gene was amplified from clethidem-resistant and susceptible plants, then sequenced to identify previously unknown mutations. Amplifications of ACCase were performed using 300ng of genomic DNA and GoTaq® Green Master Mix. The Wizard® SV Gel and PCR Clean-Up System was used to purify PCR products directly or from agarose gels.
(0003721) |
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Products: GoTaq® Green Master Mix | Wizard® SV Gel and PCR Clean-Up System |
| 15. |
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 |
| 16. |
Miozzo, M.C., Maxzud, M.K., Pacharoni, C.M., Mutal, S.A. and Modesti, N.M.
(2007)
Characterization of the variant allele 9.2 of Penta D locus.
J. Forensic Sci.
52
,
1073–6
.
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| |
Notes:
During casework analysis using the PowerPlex® 16 System, the authors identified an off-ladder allele at the Penta D locus as the microvariant allele 9.2. DNA from individuals with the 9.2 allele was amplified using a single primer pair specific for Penta D, and the amplification products were purified and sequenced to characterize the microvariant allele. PCR products were purified using the Wizard® PCR Preps DNA Purification System. Sequence analysis revealed that the 9.2 allele has 10 STR repeats and a TAA deletion in the 3´ flanking region.
(0003771) |
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Products: PowerPlex® 16 System | Wizard® PCR Preps DNA Purification System |
| 17. |
Treeck, O., Pfeiler ,G., Horn, F., Federhofer, B., Houlihan, H., Vollmer, A., and Ortmann, O.
(2007)
Novel estrogen receptor beta transcript variants identified in human breast cancer cells affect cell growth and apoptosis of COS-1 cells.
Mol. Cell. Endocrinol.
264
,
50-60
.
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| |
Notes:
This study identified two novel transcript variants of the estrogen receptor ERβ that were expressed in the ERα-negative breast cancer cell line MDA-MD-231. These variants were identified after amplification of ERβ transcripts from the breast cancer cell line by RT-PCR. The amplification products were then excised from gels and subcloned into the pTARGET™ Mammalian Expression Vector prior to sequencing. COS1 cells, which do not express the estrogen receptor, were then stably transfected with full-length ERβ or one of the splice variants and the effects on cell proliferation, apoptosis, and estrogen response were evaluated. In COS1 cells expressing either ERβ or the transcript variants cell proliferation decreased and basal apoptosis (caspase 3/7 activity) increased, compared to cells transfected with vector alone. Exposure to therapeutic doses of tamoxifen induced apoptosis in cells expressing the full-length ERβ but not in cells expressing either of the variant isoforms.
(0003618) |
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Products: Caspase-Glo® 3/7 Assay | CellTiter-Blue® Cell Viability Assay | ImProm-II™ Reverse Transcriptase | M-MLV Reverse Transcriptase Buffer Pack | M-MLV Reverse Transcriptase, RNase H Minus | pTARGET™ Mammalian Expression Vector System |
| 18. |
De Maesschalck, K,. Vanhoutte, E., Knaepen, K., Vanderheyden, N., Cassiman, J.J., and Decorte, R.
(2007)
Y-chromosomal STR haplotypes in a Belgian population sample and identification of a micro-variant with a flanking site mutation at DYS19.
Forensic Sci. Int.
152
,
89–94
.
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Notes:
The authors collected DNA samples from 113 unrelated Belgian males. The PowerPlex® Y System and a GeneAmp® 9700 PCR system were used to amplify 12 Y-chromosome STR loci (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439). Amplification products were detected using an ABI PRISM® 3100 genetic analyzer and POP-6™ polymer. Allele and haplotype frequencies and haplotype diversity were calculated. A total of 99 different haplotypes were observed.
(0003655) |
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Products: PowerPlex® Y System |
| 19. |
Davoren, J., Vanek, D., Konjhodzic, R., Crews, J., Huffine, E. and Parsons, T.J.
(2007)
Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves.
Forensic Sci. Int.
48
,
478–85
.
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| |
Notes:
The authors compared two DNA extraction methods: the International Commission on Missing Persons silica method and the standard phenol:chloroform method to determine the preferred method for extraction of DNA from skeletal remains. The efficacy of DNA extraction was measured by real-time PCR to quantify DNA and to check for the presence of PCR inhibitors, and by amplification with the PowerPlex® 16 System. DNA was extracted from processed bone powder, and 10µl of the final extract was amplified using the PowerPlex® 16 System and GeneAmp® PCR System 9700 according to the manufacturer's recommendations, except that the extension time was doubled from 30 seconds to 60 seconds for the first 10 cycles and from 45 seconds to 90 seconds for the next 22 cycles. Amplified products were detected using the ABI PRISM® 3100 Genetic Analyzer. The authors concluded that the silica-based method gave better results in autosomal STR typing than the organic extraction method.
(0003818) |
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Products: PowerPlex® 16 System |
| 20. |
Narkuti, V., Vellanki, R.N., Gandhi, K.P., Doddapaneni, K.K., Yelavarthi, P.D. and Mangamoori, L.N.
(2007)
Microsatellite mutation in the maternally/paternally transmitted D18S51 locus: two cases of allele mismatch in the child.
Clinica Chimica Acta
381
,
171-175
.
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Notes:
The authors describe two cases of paternity dispute, one with a maternally mismatched allele at the D18S51 locus and a second with a paternally mismatched D18S51 allele. Seventeen autosomal STR loci were analyzed using the PowerPlex® 16 System and AmpFlSTR® Identifiler® kit. Amplifications were performed using a GeneAmp® PCR System 9700, and amplification products were detected using an ABI PRISM® 310 Genetic Analyzer. Y-STR loci and mitochondrial DNA hypervariable regions HV1 and HV2 were also examined. Sequence analysis of the D18S51 locus revealed an expansion of the maternal allele by one repeat unit in one case and an expansion of the paternal allele by two repeat units in the second case.
(0003809) |
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Products: PowerPlex® 16 System |
| 21. |
Atallah, Z.K., Bae, J., Jansky, S.H., Rouse, D.I., and Stevenson, W.R.
(2007)
Multiplex real-time quantitative PCR to detect and quantify Verticillium dahliae colonization in potato lines that differ in response to Verticillium wilt.
Phytopathology
97
,
865-872
.
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Notes:
These authors developed a quantitative, real-time PCR method for the detection of Verticillium dahliae in potato cultivars. V. dahiae is the causative agent of Verticillium wilt, also known as potato early dying (PED). The standard detection method is a plating assay that takes 2 weeks to complete. The authors of this study performed qPCR assays using the V. dahliae beta tubulin-2 gene as a target. Initially, they amplified, subcloned and sequenced seven different genes in order to identify targets that were polymorphic among the genus Verticillium, but monoprphic in V. dahliae. After selection of beta-tubulin 2 as a suitable target, monoplex and duplex qPCR assays were performed using a Bio-Rad iCycler thermal cycler and 1ng DNA from various cultivars. For the duplex assays, the Plexor® qPCR System was used to amplify both beta-tubulin 2 and beta-actin genes simultaneously. One beta-tubulin primer was labeled with FAM, and one actin primer was labeled with Redmond Red phopsphoramidite. Amplifications were performed in 25µl reactions with 200nM each primer, 1ng DNA, and the Plexor® Master Mix. Cycling conditions were as follows: 2 minutes at 95°C; 40 cycles of 5s at 95°C, 35s at 61°C. Melt curve analysis was performed to confirm the specificity of the amplification products. The qPCR assays were shown to be faster and more sensitive than the standard plating technique, and were one order of magnitude more sensitive than other PCR-based assays.
(0003673) |
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Products: PCR Master Mix | Plexor® qPCR System |
| 22. |
Tindall, E.A., Speight, G., Petersen, D.C., Padilla, E.J., and Hayes, V.M.
(2007)
Novel Plexor SNP genotyping technology: comparisons with TaqMan and homogenous MassEXTEND MALDI-TOF mass spectrometry.
Human Mutation
0
,
1-6
.
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Notes:
This study compared the performance of the Plexor® qPCR System with the TaqMan® and MassEXTEND™ methods for genotyping analysis of 11 SNPs in >2000 DNA samples. All three methods were shown to be equivalent in call rate and accuracy. The Plexor® System is described as a cost-effective, efficient alternative to the TaqMan® technology for medium-throughput SNP analysis. Plexor® qPCR System reactions contained 5ng template DNA, 0.2µl 5µM allele-specific primers, 0.2µl 10µM anchor primer, and 2.5µl 2X Plexor™ Master Mix in a total volume of 5µl. PCR was performed on an ABI PRISM® 7900HT Sequence Detection System using the following cycling conditions: 95°C for 2 minutes; 50°C for 35s; and 40 cycles of 95°C for 5s, 60°C for 35s. Primers were designed using the Plexor® Primer Design Software.
(0003674) |
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Products: Plexor® qPCR System |
| 23. |
Kanayama, T., Arito, M., So, K., Hachimura, S., Inoue, J. and Sato, R.
(2007)
Interaction between sterol regulatory element-binding proteins and liver receptor homolog-1 reciprocally suppresses their transcriptional activities.
J. Biol. Chem.
282
,
10290–10298
.
|
| |
Notes:
To explore the interaction of liver receptor homolog (LRH)-1, a known suppressor of sterol regulatory element-binding protein (SREBP) transcriptional activity, human LRH-1 was reverse transcribed then amplified by PCR from total RNA from HepG2 cells. The amplification product was ligated into the pTargeT™ Mammalian Expression Vector to create pTarget-LRH1. For reporter experiments, a PCR fragment that encompassed the 1.3kb 5’-promoter region of the human small heterodimer partner (SHP) gene was cloned into the pGL3-Basic Vector (designated pSRB). The pGL3-Promoter Vector was used to construct pLRHREx3, which contains three LRH-1 response elements, and the insert was generated using synthetic oligonucleotides. HEK293 cells were cotransfected with 0.2µg of a promoter-firefly luciferase construct, 0.1µg of a SREBP expression plasmid, 10ng of phRL-TK Vector and 0.2 or 0.6µg of pTarget-LRH1. Alternatively, the cotransfected plasmids were 0.2µg of pSHP, 0.1µg of pTarget-LRH1, 10ng of phRL-TK Vector and 0.2 or 0.6µg of a SREBP expression plasmid. The pLRHREx3 construct (0.2µg) was cotransfected with 0.1µg of a LRH-1 expression plasmid, 0.2µg of pCMXPGC-1α (peroxisome proliferator activated receptor γ coactivator-1α), 10ng of phRL-TK Vector, and 0.1 or 0.3µg of a pSREBP expression vector in HEK 293 cells. Luciferase expression was assayed 48 hours post-transfection using the Dual-Luciferase® Assay Reporter System. To express SREBPs and LRH-1 in vitro, inserts were ligated into the pTNT™ Vector, synthesized using the TNT® Coupled Transcription/Translation System with radiolabeled methionine. Ten microliters of the 35S-labelled protein was then used in a GST-pulldown assay.
(0003692) |
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Products: Dual-Luciferase® Reporter 1000 Assay System | Dual-Luciferase® Reporter Assay System | Dual-Luciferase® Reporter Assay System 10-Pack | pGL3-Basic Vector | pGL3-Promoter Vector | pTNT™ Vector | TNT® SP6 Coupled Reticulocyte Lysate System | TNT® SP6 Coupled Reticulocyte Lysate System, Trial Size | TNT® T3 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System | TNT® T7 Coupled Reticulocyte Lysate System, Trial Size |
| 24. |
Ellison, T.I., Eckert, R.L. and MacDonald, P.N.
(2007)
Evidence for 1,25-dihydroxyvitamin D3-independent transactivation by the vitamin D receptor: uncoupling the receptor and ligand in keratinocytes.
J. Biol. Chem.
282
,
10953–10962
.
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| |
Notes:
While the absence of the Vitamin D receptor (VDR) has profound effects in skin cells, mutation of 25-hydroxyvitamin D 1α-hydroxylase (24OHase), the enzyme required for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone biosynthesis, has little effect on the skin. To determine how VDR may transactivate independent of the 1,25(OH)2D3 ligand, the human 24-hydroxylase promoter was amplified from MCF-7 genomic DNA, digested with XhoI and HindIII and inserted into the pGL3-Basic Vector. Mutations in the proximal and distal vitamin D response elements in the human 24-hydroxylase promoter were introduced using the GeneEditor™ Site-Directed Mutagenesis System. HaCaT cells, primary human fibroblasts or primary human keratinocytes were seeded at a density of 3.2 × 104 cells/well in 12-well plates and transiently transfected with reporter constructs. After 18 hours, the cells were exposed to 1,25(OH)2D3, 9-cis-retinoic acid, ethanol vehicle, or no additive and harvested 24 hours later. The luciferase activity of the cell lysates was measured using the Dual-Luciferase® Reporter Assay System. Five micrograms of RNA purified from mouse keratinocyte and fibroblast cultures was reverse transcribed and amplified for the 24OHase transcripts using the PCR Master Mix. The products were analyzed on ethidium bromide-stained 2% agarose gels.
(0003695) |
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| 25. |
Renaud, S., Loukinov, D., Abdullaev, Z., Guilleret, I., Bosman, F.T., Lobanenkov, V. and Benhattar, J.
(2007)
Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene
Nucleic Acids Research
35
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1245-1256
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Notes:
Telomeres shorten by 50–100 bases with each cell division, making the telomere a "mitotic counter" that can limit cellular lifespan. Telomerase is a two-component protein consisting of a reverse transcriptase (hTERT) bound to its own RNA template that can act to maintain telomere length in dividing cells. Telomerase is highly active in dividing cells such as germ cells, stem cells and many cancers. This paper investigated the role of methylation of the hTERT promoter and the transcription factor CTCF in regulation of telomerase activity. LacZ reporter plasmids driven by the hTERT minimal promoter were transiently transfected into HeLa cells, and reporter assays were performed on lysate generated using Passive Lysis Buffer. The hTERT minimal promoter did not show activity if all of the CpG sites were methylated. The promoter and first exon of hTERT were amplified using PCR Master Mix from sodium bisulfite-treated genomic DNA isolated from telomerase-positive cell lines and tissues. The resulting fragments were cloned using the pGEM®-T Vector System II. For the methylation cassette assay, methylated and unmethylated fragments were cloned into a methylated or unmethylated vector using the LigaFast™ Rapid DNA Ligation System. The authors conclude that methylation plays a dual role in regulating hTERT expression. CTCF will bind to the first exon of hTERT when the hTERT CpG island is not methylated, resulting in downregulation of hTERT expression. Although CTCF cannot bind the hTERT promoter when the DNA is completely methylated, the methylation itself completely represses transcription. In situations where there is partial methylation of the promoter, such as in tumor cells, CTCF cannot bind to the promoter, but the partial methylation is not enough to repress transcription, and hTERT is expressed.
(0003641) |
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