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Microbiology 153, 3023–3033. Expression analysis of extracellular proteins from Phanerochaete chrysosporium grown on different liquid and solid substrates. 2007

Sato, S., Liu, F., Koc, H. and Tien, M.

Notes: The authors characterized expression of extracellular proteins by white-rot fungus, Phanerochaete chrysosporium, grown on wood. Temporal expression of these proteins was monitored by relative quantitative RT-PCR. Two micrograms of total RNA was reversed transcribed using 1µg of Random Primers at 37°C for 1 hour. PCRs with one set of PCR primers were performed using 0.5 units of GoTaq® DNA Polymerase, 1X reaction buffer, 250µM each dNTP, 0.5µM each primer and 1µl of cDNA. PCRs with two sets of PCR primers were performed using 2.5 units of GoTaq® DNA Polymerase, 1.6X reaction buffer, 500µM each dNTP, 0.5µM each primer and 1µl of cDNA. (3708)

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J. Biol. Chem. 282, 14364–14372. Expression of sialidase Neu2 in leukemic K562 cells induces apoptosis by impairing Bcr-Abl/Src kinases signaling. 2007

Tringali, C., Lupo, B., Anastasia, L., Papini, N., Monti, E., Bresciani, R., Tettamanti, G. and Venerando, B.

Notes: The authors transfected the myleoid leukemic cell line K562 with the cytosolic sialidase Neu2. Expression of Neu2 resulted in a significant decrease in mRNA levels for the anti-apoptotic factors Bcl-XL and Bcl-2 as determined by real-time PCR. Reverse transcription was carried out with 1µg of total RNA using the ImProm-II™ Reverse Transcription System and random hexamers. cDNA representing 10ng of total RNA was used in a real-time PCR to quantitate Bcl-XL and Bcl-2 mRNA levels. (3725)

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Appl. Environ. Microbiol. 74, 811-817. High frequency of histamine-producing bacteria in enological environment and instability of the phenotype. 2007

Lucas, P.M., Claisse, O. and Lonvaud-Funel, A.

Notes: Because of concerns about the consumption of histamine in wine (which makes histamine more potent), the quantity of lactic acid bacteria (LAB), which can produce histamine during winemaking, was determined in 264 samples of red wine from 116 wineries. DNA was isolated from LAB strains grown in De Man Rogosa Sharpe (MRS) broth using the Wizard® Genomic DNA Purification Kit. Glass beads were used to disrupt the cells, and a modified Wizard® Genomic DNA Purification protocol that included PVP treatment was performed. The purified DNA was used in qPCR analysis. (3741)

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Forensic Sci. Int. 48, 478–85. Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves. 2007

Davoren, J., Vanek, D., Konjhodzic, R., Crews, J., Huffine, E. and Parsons, T.J.

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. (3818)

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J. Clin. Invest. 117, 3042–3048. HLA class I polymorphisms are associated with development of infectious mononucleosis upon primary EBV infection. 2007

McAulay, K.A., Higgins, C.D., Macsween, K.F., Lake, A., Jarrett, R.F., Robertson, F.L., Williams, H. and Crawford, D.H.

Notes: The authors examined whether genetic differences at the HLA class I locus affect development of Epstein Barr Virus-associated diseases. Peripheral blood mononuclear cells were isolated from asymptomatic EBV-seropositive and seronegative individuals and patients with acute infectious mononucleosis. DNA was isolated, and genotypes at two HLA class I loci and one HLA class III locus, as a control, were determined by PCR. The 10µl PCRs contained 25ng of DNA, 1X GoTaq® Flexi Reaction Buffer, 2.5mM MgCl2, 200µM dNTP, 0.5 units of GoTaq® Flexi DNA Polymerase and 25µM of forward and reverse primer, one of which was labeled with 6-FAM fluorescent dye. The results show that HLA class I polymorphisms might predispose people to develop infectious mononucleosis upon EBV infection. (3712)

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Emerging Infect. Dis. 13, 1756-1758. Human Bocavirus infection in children with gastroenteritis, Brazil. 2007

Albuquerque, M.C., Rocha, L.N., Benati, F.J., Soares, C.C., Maranhão, A.G., Ramírez, M.L, Erdman, D., and Santos, N.

Notes: In this study, the Wizard® Genomic DNA Purification Kit was used to extract DNA from diluted fecal samples. The extracted DNA was used in PCR with specific primers to detect viral sequences. PCR fragments were gel-purified using the SV Gel and PCR Clean-Up System prior to sequencing to confirm the Bocavirus DNA identity. (4221)

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J. Mol. Endocrinol. 36, 449–461. Human chorionic gonadotropin-dependent induction of an equine aldo-keto reductase (AKR1C23) with 20alpha-hydroxysteroid dehydrogenase activity during follicular luteinization in vivo. 2007

Brown, K.A., Boerboom, D., Bouchard, N., Doré, M., Lussier, J.G. and Sirois, J.

Notes: The authors cloned the novel equine aldo-keto reductase AKR1C23 and characterized its expression patterns in the preovulatory follicle. The AKR1C23 cDNA was amplified from equine ovarian RNA using the Access RT-PCR System and primers designed by sequence alignments of known AKR sequences, then cloned into the pGEM®-T Easy Vector. Levels of AKR1C23 and ribosomal protein L17a mRNAs in various equine tissues were quantified using the Access RT-PCR System and 21 cycles and 18 cycles, respectively, followed by agarose gel electrophoresis, transfer to nylon membranes, and hybridization to radiolabeled probes synthesized using the Prime-a-Gene® Labeling System. (3791)

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J. Immunol. 178, 986-92. Identification of CXCL11 as a STAT3-dependent gene induced by IFN. 2007

Yang, C.H., Wei, L., Pfeffer, S.R., Du, Z., Murti, A., Valentine, W.J., Zheng, Y. and Pfeffer, L.M.

Notes: The STAT proteins are involved in the transcriptional response to interferon (IFN), which includes induction of CXCL11 and ISG15 genes. The authors used quantitative real-time PCR to examine CXCL11 and ISG15 expression levels in IFN-sensitive and IFN-resistant cells after IFN treatment. Expression levels of the IFN-responsive genes were normalized to that of β-actin. Total RNA was isolated from untreated and IFN-treated cells, and qPCR was performed on a Bio-Rad iCycler® instrument using the AccessQuick™ RT-PCR System and SYBR® Green I. Reverse transcription was performed at 48°C for 45 minutes, followed by 35 cycles of PCR. Prior to qRT-PCR, PCR product size was confirmed by agarose gel electrophoresis, and PCR specificity was checked by analyzing melting curves. (3767)

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Nucl. Acids Res. 35, 2060–73. In vivo and in vitro investigation of bacterial type B RNase P interaction with tRNA 3'-CCA. 2007

Wegscheid, B. and Hartmann, R.K.

Notes: The authors used RT-PCR to quantitate the level of rnpB expression after complementing the Bacillus subtilis rnpB mutant strain SSB318 with wildtype or mutant rnpB genes from S. aureus or B. subtilis. Preliminary experiments with decreasing amounts of RNA template were performed to show that rnpB RT-PCR product yields were linearly dependent on RNA template amounts after 12 PCR cycles. RT-PCR was performed using the Access RT-PCR System. (3794)

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J. Virol. 81, 173–81. In vivo packaging of brome mosaic virus RNA3, but not RNAs 1 and 2, is dependent on a cis-acting 3' tRNA-like structure. 2007

Annamalai, P. and Rao, A.L.

Notes: The brome mosaic virus (BMV) contains four RNAs (B1, B2, B3 and B4), each of which includes a tRNA-like structure (TLS) that is required for packaging in vitro. To confirm the necessity of the TLS for packaging in vivo, the authors created TLS-less B1, B2 and B3 RNAs; TLS-less B1 and B2 RNAs were packaged, while the TLS-less B3 RNA was not. Co-infiltration of Nicotiana benthamiana leaves with wildtype B1 and B2 and TLS-less B3 RNA resulted in restoration of the B3 TLS. To determine whether the resulting TLS was regained by homologous or heterologous recombination with B1 or B2 RNA, the 3´ end of the B3 RNA was amplified from leaf total RNA by RT-PCR using the AccessQuick™ RT-PCR System, then sequenced. (3768)

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J. Biol. Chem. 282, 10290–10298. Interaction between sterol regulatory element-binding proteins and liver receptor homolog-1 reciprocally suppresses their transcriptional activities. 2007

Kanayama, T., Arito, M., So, K., Hachimura, S., Inoue, J. and Sato, R.

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. (3692)

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Cancer Res. 67, 10600–10607. Interaction of the tumor metastasis suppressor nonmetastatic protein 23 homologue H1 and estrogen receptor alpha alters estrogen-responsive gene expression. 2007

Curtis, C.D., Likhite, V.S., McLeod, I.X., Yates, J.R. and Nardulli, A.M.

Notes: Tumor metastasis suppressor nonmetastatic protein 23 homologue 1 (NM23-H1) interacts with estrogen receptor α (ERα) and influences ERα-mediated gene expression. The authors knocked down NM23-H1 expression using RNA interference in estrogen-treated or untreated MC-7 human breast cancer cells and determined the effect on transcription of estrogen-responsive genes, including progesterone receptor, Bcl-2, cathepsin D and cyclin D1. Levels of these mRNAs were measured in the presence of NM23-H1 or control small interfering RNAs using quantitative RT-PCR. Total RNA was treated with RQ1 RNase-Free DNase to remove contaminating DNA, and cDNA was synthesized using the Reverse Transcription System. The resulting cDNA was subjected to quantitative PCR using SYBR® Green dye. (3789)

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FASEB J. 21, 1893–1901. Low expression of COX-2, reduced cumulus expansion, and impaired ovulation in SULT1E1-deficient mice. 2007

Gershon, E., Hourvitz, A., Reikhav, S., Maman, E. and Dekel, N.

Notes: The authors investigated the role of estrogen inactivation by the SULT1E1-encoded estrogen sulfotransferase in ovulation in mice. Semiquantitative RT-PCR was used to characterize the temporal and tissue-specific expression of SULT1E1 mRNA in ovulating mice. First-strand cDNA synthesis was performed at 37°C for 2 hours using 7.5µg of total RNA, 1µl (0.5µg) of oligo(dT)15, 40 units of RNasin® Ribonuclease Inhibitor and 200 units of M-MLV Reverse Transcriptase. (3913)

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Proc. Natl. Acad. Sci. USA 104, 12796-12800. Metabolic plasticity during mammalian development is directionally dependent on early nutritional status. 2007

Gluckman PD, Lillycrop KA, Vickers MH, Pleasants AB, Phillips ES, Beedle AS, Burdge GC, Hanson MA.

Notes: To examine how exposure to leptin in utero affects hepatic gene expression and epigenetic status in adulthood, pregnant Wistar rats were fed a standard diet or 30% undernutrition. The three-day-old pups were exposed to saline or leptin for 10 days, weaned and either fed a standard or high-fat diet. On day 170, the rats were sacrificed and tissues snap frozen. Five micrograms of genomic DNA was isolated from rat liver using the Wizard® SV Genomic DNA Purification System. Then 25ng of the purified DNA was digested with methylation-sensitive restriction enzymes, and the glucocorticoid receptor (GR) and peroxisome proliferator-activated receptor alpha (PPARα) fragments were amplified by real-time PCR. (3744)

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Clinica Chimica Acta 381, 171-175. Microsatellite mutation in the maternally/paternally transmitted D18S51 locus: two cases of allele mismatch in the child. 2007

Narkuti, V., Vellanki, R.N., Gandhi, K.P., Doddapaneni, K.K., Yelavarthi, P.D. and Mangamoori, L.N.

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. (3809)

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Brain Res. 1127, 66–75. Molecular characterization and gene expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) in the lizard brain. 2007

Valiante, S., Prisco, M., Capaldo, A., Zambrano, I., De Falco, M., Andreuccetti, P., Laforgia, V., and Varano, L.

Notes: The authors cloned pituitary adenylate cyclase-activating polypeptide (PACAP) from lizard (Podarcis sicula) brain. They then isolated total RNA from lizard brain using the SV Total RNA Isolation System and used 4µg of total RNA in a reverse transcription with ImProm-II™ Reverse Transcriptase and oligo(dT)15 primers at 37°C for 1.5 hours. The PACAP cDNA was amplified by PCR, and the resulting PCR products were cleaned up using the Wizard® SV Gel and PCR Clean-Up System prior to sequencing. (3666)

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Nucl. Acids Res. 35, 2390–2402. Molecular mechanism of upregulation of survivin transcription by the AT-rich DNA-binding ligand, Hoechst33342: evidence for survivin involvement in drug resistance. 2007

Wu, J., Apontes, P., Song, L., Liang, P., Yang, L. and Li, F.

Notes: To study how Hoechst33342 upregulates the expression and promoter activity of survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, nested deletions of the survivin promoter driving a firefly luciferase reporter gene (pLuc-1430c ) were created using the Erase-a-Base® System. The vector was digested with SalI, the ends filled in using α-phosphorothioate dNTPs, digested a second time with BamHI and subjected to Exonuclease III digestion at 25°C. Aliquots of the 5’ end deletions were removed every 15–30 seconds, religated, transformed and analyzed by PCR and sequencing. Transient transfection experiments were carried out using HeLa cells seeded in 24-well plates and cotransfected 490ng of a pLuc-survivin construct and 10ng of pRL-TK Vector or in U937 cells using 2µg of survivin promoter constructs. After 24 hours, the HeLa cells were treated with Hoechst33342 and harvested 8–24 hours later. For U937 cells, the medium was changed with or without added drugs and the cells lysed after 36 hours. Reporter expression was assessed using the Dual-Luciferase® Reporter Assay System. (3697)

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J. Clin. Microbiol. 45, 1469–1477. Multilocus sequence typing of the pathogenic fungus Aspergillus fumigatus. 2007

Bain, J.M., Tavanti, A., Davidson, A.D., Jacobsen, M.D., Shaw, D., Gow, N.A. and Odds, F.C.

Notes: The authors developed a multilocus sequence typing scheme (MLST) to examine sequence variation and discriminate between Aspergillus fumigatus strains. They also examined the distribution of MAT1-1 and MAT1-2 sexual idiomorphs in 100 clinical and environmental isolates. Sexual idiomorphs were determined using PCR and a reverse primer to both idiomorphs and a forward primer specific to either MAT-1 or MAT-2. PCRs consisted of 2mM MgCl2, 200µM DNTPs and 2.5 units of GoTaq® DNA Polymerase. (3714)

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Phytopathology 97, 865-872. Multiplex real-time quantitative PCR to detect and quantify Verticillium dahliae colonization in potato lines that differ in response to Verticillium wilt. 2007

Atallah, Z.K., Bae, J., Jansky, S.H., Rouse, D.I., and Stevenson, W.R.

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. (3673)

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J. Endocrinol. 197, 201–12. Neonatal exposure to bisphenol A modifies the abundance of estrogen receptor alpha transcripts with alternative 5'-untranslated regions in the female rat preoptic area. 2007

Monje, L., Varayoud, J., Luque, E.H. and Ramos, J.G.

Notes: The authors investigated the effect of neonatal bisphenol A (BPA) exposure in rats on expression of estrogen receptor α (ERα) transcripts. Alternative ERα transcripts in preoptic area of treated and untreated rats were quantified using real-time RT-PCR. Reverse transcription was performed using 4µg of total RNA, 200pmol random primers and 300 units M-MLV Reverse Transcriptase. Real-time PCR was performed using SYBR® Green I to quantify amplified products. To determine if the changes in BPA-induced ERα transcript expression were caused by DNA methylation, the methylation status of the five ERα promoters was examined by bisulfite modification. Genomic DNA was isolated from rat tissue using the Wizard® Genomic DNA Purification Kit, denatured with NaOH, then treated with hydroquinone and sodium bisulfite. Prior to methylation-specific PCR, DNA was cleaned up using the Wizard® DNA Purification Resin as directed by the manufacturer. PCR products were cleaned up again using the Wizard® SV Gel and PCR Clean-Up System, then subjected to restriction enzyme digestion and agarose gel electrophoresis to reveal methylation-dependent sequence differences. (3911)

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Mol. Cell. Endocrinol. 264, 50-60. Novel estrogen receptor beta transcript variants identified in human breast cancer cells affect cell growth and apoptosis of COS-1 cells. 2007

Treeck, O., Pfeiler ,G., Horn, F., Federhofer, B., Houlihan, H., Vollmer, A., and Ortmann, O.

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. (3618)

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Hum. Mutat. 0, 1-6. Novel Plexor SNP genotyping technology: comparisons with TaqMan and homogenous MassEXTEND MALDI-TOF mass spectrometry. 2007

Tindall, E.A., Speight, G., Petersen, D.C., Padilla, E.J., and Hayes, V.M.

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. (3674)

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J. Biomol. Scr. 12, 546–559. Optimization procedure for small interfering RNA transfection in a 384-well format. 2007

Borawski, J., Lindeman, A., Buxton, F., Labow, M. and Gaither, L.A.

Notes: A lentiviral expression vector containing the firefly luciferase gene from a pGL3 Vector was transduced into SKOV3 cells in 384-well plates, transfected with various siRNAs and analyzed 72 hours later. The luciferase expression was determined using the Bright-Glo™ Luciferase Assay System and cell viability assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. (3729)

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Can. J. Vet. Res. 71, 230–235. Presence of non-O157 Shiga toxin-producing Escherichia coli in feces from feedlot cattle in Alberta and absence on corresponding beef carcasses. 2007

Renter, D.G., Bohaychuk, V., Van Donkersgoed, J. and King, R.

Notes: Beef carcasses were tested for the presence of non-O157 Shiga toxin-producing Escherichia coli (STEC) to determine prevalence and serotypes. Carcasses were swabbed with a sponge, incubated in brain–heart infusion broth and 300µl of growth transferred to a separate tube. The bacteria were diluted in water, spun and then the DNA isolated using the Magnesil® KF, Genomic System on the Thermo Electron KingFisher® mL instrument. This extracted DNA was then tested by multiplex PCR for the Shiga toxin 1 (stx1) and Shiga toxin 2 (stx2) genes. (3762)

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J. Biol. Chem. 282, 35405-35415. Protein-tyrosine phosphatase H1 controls growth hormone receptor signaling and systemic growth. 2007

Pilecka, I., Patrignani, C., Pescini, R., Curchod, M.L., Perrin, D., Xue, Y., Yasenchak, J., Clark, A., Magnone, M.C., Zaratin, P., Valenzuela, D., Rommel, C. and van Huijsduijnen, R.H.

Notes: To genotype PTPH1 knock-out (KO), heterozygous (HET), and wild type (WT) mice, tail snips were digested overnight with proteinase K and the DNA trapped using the Wizard® SV 96 Genomic DNA Purification System. Genomic DNA was washed using the Wizard® SV Wash Solution and eluted in 200μl of water at 65°C. The protease was inactivated at 95°C and 2μl of DNA was used for PCR. (3739)

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