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Assay Drug Dev. Technol. 5, 127–136. Bioluminescent assays for high-throughput screening 2007

Fan, F. and Wood, K.V.

Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)

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J. Biol. Chem. 282, 9883–94. Cell confluence-induced activation of signal transducer and activator of transcription-3 (Stat3) triggers epithelial dome formation via augmentation of sodium hydrogen exchanger-3 (NHE3) expression. 2007

Su, H.W., Yeh, H.H., Wang, S.W., Shen, M.R., Chen, T.L., Kiela, P.R., Ghishan, F.K. and Tang, M.J

Notes: The authors tested their hypothesis that Na+-H+ exchangers (NHE) are involved in the formation of multicullar dome structures in confluent Madin-Darby canine kidney (MDCK) cells and that the Stat3 pathway is involved in regulation of NHEs. The authors performed semi-quantitative RT-PCR to monitor NHE3 mRNA levels in MDCK cells expressing a constitutive Stat3 mutant or a dominant-negative Stat3 mutant. The reverse transcription step was performed using Promega M-MLV Reverse Transcriptase. RAlso, Stat3 activities in low-density cultures and high-density cultures were compared using a reporter gene assay. Four copies of the Stat3-binding site were cloned upstream of a firefly luciferase reporter gene, and the resulting vector, along with the pRL-TK Vector for normalization, were transfected into MDCK cells. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System. (3910)

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J. Forensic Sci. 52, 1073–6. Characterization of the variant allele 9.2 of Penta D locus. 2007

Miozzo, M.C., Maxzud, M.K., Pacharoni, C.M., Mutal, S.A. and Modesti, N.M.

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. (3771)

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Proc. Natl. Acad. Sci. USA 104, 10637–10642. Commensal and pathogenic Escherichia coli use a common pilus adherence factor for epithelial cell colonization. 2007

Rendón, M.A., Saldaña, Z., Erdem, A.L., Monteiro-Neto, V., Vázquez, A., Kaper, J.B., Puente, J.L. and Girón, J.A.

Notes: The authors identified an adherence factor of enterohemorrhagic E. coli that is involved in colonization of cultured epithelial cells. This factor, named E. coli common pilus (ECP), is encoded by the ecpA gene, which is present 96% of E. coli strains tested, as determined by PCR. The remaining 4% of the strains were found to be deficient in the ECP operon, as determined by multiplex PCR amplification of ecpR, ecpA, epcB and ecpC sequences. PCR were performed using GoTaq® Green Master Mix. An ecpA deletion mutant exhibited impaired adherence compared to the wildtype E. coli strain. Complementation of the mutant strain with the plasmid pMR13, the pGEM®-T Vector cotaining the ecpA gene, restored the strain's ability to adhere to epithelial cells. (3719)

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J. Forensic Sci. 52, 870–3. Concordance study between the AmpFlSTR MiniFiler PCR amplification kit and conventional STR typing kits. 2007

Hill, C.R., Kline, M.C., Mulero, J.J., Lagacé, R.E., Chang, C.W., Hennessy, L.K. and Butler, J.M.

Notes: The authors analyzed 1,308 samples for concordance between the Identifiler® kit, AmpFlSTR® Minifiler™ kit and PowerPlex® 16 System. DNA was isolated from liquid blood using the manual DNA IQ™ System protocol, and STR amplifications were performed as per the manufacturer's recommendations except that reaction volumes were decreased by half. Amplified products were analyzed using an Applied Biosystems 3130xl and POP™-4 or POP™-6 polymer. Twenty seven disconcordant phenotypes were identified between the Minifiler™ and Identifiler® kits, 14 between Minifiler™ and PowerPlex® 16 kits, and 4 between PowerPlex® 16 and Identifiler® kits. (3770)

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J. Biol. Chem. 282, 21798–21809. Control and regulation of KplE1 prophage site-specific recombination: a new recombination module analyzed. 2007

Panis, G., Méjean, V. and Ansaldi, M.

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. (3722)

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J. Biol. Chem. 282, 21818–21828. Degradation of hsp70 and other mRNAs in Drosophila via the 5´ 3´ pathway and its regulation by heat shock. 2007

Bönisch, C., Temme, C., Moritz, B. and Wahle, E.

Notes: The authors studied hsp70 mRNA degradation in Drosophila Schneider cells. mRNA deadenylation and decay were monitored by Northern blot. Two of the Northern blot probes used to visualize the mRNA decay products were synthesized by transcription of linearized plasmids using T7 RNA Polymerase and [α-32P] UTP. A population of deadenylated mRNA was created by hybridizing mRNA with oligo(dT) and treating with RNase H. CCR4•NOT was identified as the main deadenylase involved in mRNA decay, and the PAN2:PAN3 deadenylase was a minor contributor. RNA interference was used to knock down expression of PAN2 and CAF1, a subunit of CCR4•NOT, to assess the effect on mRNA decay. Reduced expression levels of PAN2 and CAF1 were confirmed by semi-quantitative RT-PCR and Western blotting, respectively. RT-PCR was performed using 1.5µg total RNA and 150 units of MMLV Reverse Transcriptase in a 25µl reaction. One microliter of the RT reaction was used as a template in an 80µl PCR using 0.5 units of GoTaq® DNA Polymerase, 1.5mM MgCl2 and 1X Green GoTaq® Flexi Reaction Buffer. (3707)

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Clin. Chem. 53, 1808–1813. Development of a novel immunoassay for the assessment of plasma Gas6 concentrations and their variation with hormonal status. 2007

Clauser, S., Peyrard, S., Gaussem, P., Crespin, M., Emmerich, J., Aiach, M. and Borgel, D.

Notes: To examine the effects of hormones on Gas6, a plasma vitamin K-dependent protein that may play a role in cardiovascular disease, the authors developed an ELISA test for Gas6, which they tested on blood from male and female volunteers. A recombinant Gas6 control was developed by reverse transcribing the full-length human Gas6 mRNA from human umbilical vein endothelial cells, amplifying the cDNA using nested PCR and after restriction digestion, ligating the insert into the EcoRI and XbaI sites of the pCI-neo Mammalian Expression Vector. The full-length construct was confirmed by sequencing and then tested in the Gas6 ELISA. (3688)

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J. Biol. Chem. 282, 29847–29854. Differential regulation of vitamin D receptor (VDR) by the p53 family: p73-dependent induction of VDR upon DNA damage. 2007

Kommagani, R., Payal, V. and Kadakia, M.P.

Notes: The authors examined transcriptional regulation of the vitamin D receptor (VDR) by p53 and p63, a member of the p53 family, under stressed and unstressed conditions. Reporter constructs with the full-length and minimal VDR promoters controlling expression of firefly luciferase were cotransfected with p53 or p63 expression constructs, and transcriptional activation of the VDR promoter was monitored using the Dual-Luciferase® Reporter 100 Assay System. Results were normalized to Renilla luciferase activity. Interaction between p73, another member of the p53 family, and the VDR promoter was examined using chromatin immunoprecipitation. The imunnopreciptated chromatin was reverse crosslinked, DNA was eluted and VDR and p21 sequences were detected by PCR using GoTaq® Green Master Mix. (3715)

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Am. J. Pathol. 171, 1153-1167. Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy. 2007

Bodó, E., Tobin, D.J., Kamenisch, Y., Bíró, T., Berneburg, M., Funk, W. and Paus, R.

Notes: To study how chemotherapy affects hair follicles (HF), the researchers microdissected and cultured human anagen VI scalp follicles and treated the cells with 4-Hydroperoxycyclophosphamide (4-HC) at similar concentrations to those received during therapy. After incubation for 24 hours with 30µmol/L of 4-HC, the HFs were homogenized and genomic DNA was purified using the Wizard® SV Genomic DNA Purification System. To determine if the treatment induced the mitochondrial common DNA deletion, the isolated DNA was subjected to real-time PCR. Further examination of gene expression was performed by isolating total RNA from two HF samples, reverse transcribing 3µg of the RNA using 15U of AMV Reverse Transcriptase and 0.025 µg/µl random primers, and amplifying seven human genes in a TaqMan® Assay. (3746)

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Plant Physiol. 145, 547–558. Diversity of acetyl-coenzyme A carboxylase mutations in resistant Lolium populations: Evaluation using clethodim. 2007

Yu, Q., Collavo, A., Zheng, M.Q., Owen, M., Sattin, M. and Powles, S.B.

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. (3721)

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Genome Res. 17, 1286-1295. Domain-wide regulation of gene expression in the human genome. 2007

Gierman, H.J., Indemans, M.H., Koster, J., Goetze, S., Seppen, J., Geerts, D., van Driel, R. and Versteeg, R.

Notes: The authors explored the possibility of a domain-based level of gene expression regulation for chromosomes by integrating the green fluorescent protein (GFP) reporter in 90 different locations in cultured human cells. This integration was accomplished by infecting human embryonic kidney cells (HEK293) with a lentiviral construct carrying the GFP gene under the control of the ubiquitously expressed human phosphoglycerate kinase (PGK), sorting GFP-positive cells by FACS and selecting clones for expansion. Genomic DNA was isolated from the various clones using the Wizard® SV Genomic DNA Purification System and analyzed by PCR or restriction digested for Southern blotting. (3743)

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Nucl. Acids Res. 35, 1245-1256. Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene 2007

Renaud, S., Loukinov, D., Abdullaev, Z., Guilleret, I., Bosman, F.T., Lobanenkov, V. and Benhattar, J.

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. (3641)

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J. Clin. Microbiol. 45, 3316-3322. Evaluation the Invader Assay with the BACTEC MGIT 960 System for prompt isolation and identification of Mycobacteria from clinical specimens. 2007

Ichimura, S., Nagano, M., Ito, N., Shimojima, M., Egashira, T., Miyamoto, C., Ohkusu, K., and Ezaki, T.

Notes: These authors compared standard culture conditions, DNA isolation and analysis (e.g, sequencing) with a liquid culture, DNA isolation and a homogeneous fluorescent detection system for identifying mycobacterial species. The standard DNA extraction began with a loopful (3–mm3 sphere) of bacterial colony grown on Ogawa slants that used glass beads to mechanically disrupt the cells. The resulting lysate was extracted using phenol/chloroform, and DNA purified from the aqueous phase using a robotic liquid handler AGE-96 (Biotec) and the MagneSil® Blood Genomic, Max Yield System. The DNA extractions were used in PCR and sequencing reactions. (3700)

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J. Biol. Chem. 282, 10953–10962. Evidence for 1,25-dihydroxyvitamin D3-independent transactivation by the vitamin D receptor: uncoupling the receptor and ligand in keratinocytes. 2007

Ellison, T.I., Eckert, R.L. and MacDonald, P.N.

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. (3695)

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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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