Notes: To prepare single rat NK cells for reverse transcription, the cells were sorted by flow cytometry and lysed using 0.5% NP-40, 2.2µl of 5X M-MLV Reverse Transcriptase Buffer, 0.3µl of 0.1mol/l DTT, 5.5µl DEPC-treated water and 1U of RNasin^® Plus RNase Inhibitor. After a 30-minute incubation on ice, the lysates were heated to 65°C for 90 seconds, cooled to 22°C for 3 minutes and placed back on ice. For the RT reaction, 1U RNasin^® Plus RNase Inhibitor, 60 U of M-MLV Reverse Transcriptase, 2.5mmol/l dNTPs and 100µM primers were added to these single-cell lysates. (3390)

Notes: The authors examined interindividual differences in cytochrome P450 CYP1A1 and CYP1B1 expression levels in human lymphocytes before and after exposure to dioxins and dioxin-like compounds. CYP1A1 and 1B1 mRNA levels were assessed with semi-quantitative RT-PCR using the Access RT-PCR System. (3432)

Notes: The presence or absence of intercellular adhesion genes (icaA and icaD) in various strains of Staphylococcus epidermidis isolated in medical facilities was investigated using PCR amplification and GoTaq^® DNA Polymerase. Each reaction contained 150ng of template. GoTaq^® Green Reaction Buffer was used in the PCR. (3359)

Notes: The authors investigated common variants of the APOA5 gene that have been associated with differences in plasma triglyceride (TG) levels. PCR fragments containing either the –1131T --> C promoter variant or containing both the –1131T --> C and –3G --> A promoter variants were cloned into the pGEM^®-T Vector System. The fragments were subsequently cloned into the pGL3 Basic Vector and transiently transfected into Huh7 and HepG2 cells along with the luciferase control vector, pRL-TK. The cells were lysed 48 hours after transfection and Luciferase activity was measured with the Dual-Luciferase^® Reporter Assay System. The function of the 1891T --> C variant in the 3´ UTR was tested the same way; with the exception that site-directed mutagenesis was performed to introduce the T --> C at position 1891 before the fragment was cloned into the pGL3 Basic Vector. The functionality of the Kozak sequence –3A --> G variant was determined by cloning cDNAs into the pGEM^®-7Zf Vector. Transcription/translation experiments were performed using the TNT^® Quick Coupled Transcription/Translation System and the proteins were labeled using the FluorTect™ Green_Lys System. In addition, a primer extension inhibition assay was performed using capped mRNAs generated with the Riboprobe^® System –T7 and the Ribo m⁷G Cap Analog. Ribosome binding reactions were performed using the Rabbit Reticulocyte Lysate System, Nuclease Treated. (3460)

Notes: On November 20, 2002, an explosion in a munitions facility left 7 people dead, 100 injured and 5 missing. The authors used DNA typing to identify 2 tissue samples and 19 bone samples. These samples, as well as reference samples from relatives of the missing persons, were analyzed using the PowerPlex^® 16 System. DNA was extracted using phenol:chloroform:isoamyl alcohol extraction and concentrated, then amplified using 28–30 cycles. For bone samples, the cycle number was increased to 35. The success rate was 90% (19 of 21 samples identified; 2 of 21 samples had a high degree of contamination and could not be identified). (3819)

Notes: These authors studied the effect of transgenic Bacillus thuringiensis (Bt) corn feed containing the Cry1A gene on broiler chicken performance. They also analyzed the degradation of the Cry1A(b) transgene in the digestive tract of the chickens. Blood and samples from cecum, jejunum, gizzard, and crop were collected from ten broilers per treatment (fed Bt corn versus near isogenic corn). Genomic DNA was isolated from blood samples and the intestinal contents of the crop and gizzard using the Wizard^® Genomic DNA Purification Kit. Fifty nanograms of purified DNA were then used in PCR analysis. (3678)

Notes: To rapidly characterize somatic mutations in the EGF receptor in lung cancer tissues, genomic DNA was extracted from lung biopsies using the Wizard^® SV Genomic DNA Purification System. The extracted DNA was used in a LightCycler SNP genotyping assay to determine the genotype of common loci associated with improved patient response to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. (3588)

Notes: To confirm that the rat IL-10 transgene under the control of a liver-specific promoter was expressed in mice, mouse tails were subjected to Proteinase K digestion and genomic DNA isolation using the Wizard^® SV 96 Genomic DNA Purification System. PCR analysis was used to distinguish between mouse and rat IL-10. (3555)

Notes: Immunohistochemistry, Western blotting, and real-time PCR were used to determine levels of COX-2 in papilloma and normal laryngeal tissue. Explant cultures of human normal laryngeal and papilloma cells were used to define the signaling pathways that regulate COX-2 expression and investigate the potential of targeting COX-2 as a strategy to suppress papilloma growth. To measure the effects of prostagladin E₂ (PGE₂) on cell number, papilloma cells were cultured in keratinocyte growth media containing 250 or 500 nmol/L PGE₂ or an equal volume of DMSO for 24 hours. The relative measure of viable cells was determined by the CellTiter 96™ Non-Radioactive Cell Proliferation Assay.
(3306)

Notes: To simulate isolating Salmonella typhimurium genomic DNA from various sample types, 2 to 2 × 10⁶ genome equivalents and 200µl lysis buffer were added to pelleted material from fishmeal, pig feces and chicken neck skin. After vortexing the samples, the genomic DNA was isolated using the MagneSil^® KF, Genomic System on a KingFisher^® workstation. To analyze the purified DNA, 5µl was used in real-time PCR with four replicates. (3425)

Notes: In this study, the researchers used the MagneHis™ Protein Purification System to purify recombinant, histidine-tagged Tribolium castaneum (red grain beetle) esterase from Pichia pastoris. The T. castaneum esterase gene, termed TCE, was cloned into pGAPZα A, pGAPZ B, and pPICZ B vectors and P. pastoris cultures transformed with each vector were analyzed for esterase activity. TCE yields varying from 7-80mg/L were obtained from P. pastoris cultures containing the above constructs using the MagneHis™ Protein Purification System. Specific activities of histidine-tagged TCE ranged from 4.5 to 5.7 U/mg. (3297)

Notes: To learn more about the molecular mechanism of thermostability of sucrose phosphate synthase (SPS) from Halothermothrix orenii, the gene was cloned in the pTrcHisA expression vector by PCR, transformed into E. coli and induced for 4 hours with 1mM IPTG. The cells were lysed by sonication and freeze/thaw cycles, and the bacterial lysate was cleared by centrifugation. The HisLink™ Protein Purification Resin was added to the cleared lysate and incubated for 1 hour with gentle agitation. The mixture was transferred to a chromatography column and washed with at least 50 column volumes of 20mM Tris-HCl (pH 7.5), 500mM NaCl, 10mM imidazole. Recombinant SPS was eluted in 20mM Tris-HCl (pH 7.5), 100mM NaCl, 500mM imidazole and the imidazole removed by diafiltration. The purified protein was then allowed to crystallize and used for X-ray diffraction to determine structure. (3281)

Notes: Suppression subtractive hybridization (SSH) was performed on six breast cancer tumors. The PCR products were T/A cloned, grown and isolated using the Wizard^® MagneSil^® Plasmid Purification System. The purified plasmid DNA was then sequenced using BigDye^® 3.0 Sequencing Mix and the ABI 3700 Genetic Analyzer. (3446)

Notes: Having observed that blunt 27mers had increased potency in RNAi compared to 21mers or 27mers with 3' or 5' overhangs, these authors investigated what differences may account for these changes in gene silencing activity using the same target sequence in enhanced green fluorescent protein (EGFP). For one experiment, a PCR-generated fragment of the EGFP coding region spanning sites EGFPS1 and EGFPS2 was cloned into psiCHECK™-2 Vector in both sense and antisense orientations. Also, a PCR-generated fragment of the human heterogeneous nuclear ribonucleoprotein H (hnRNPH) coding region spanning sites H1 and H3 was similarly cloned in sense and antisense orientations. HEK293 cells were transfected with 150ng EGFP sense and antisense vectors plus EGFPS2 or control duplex RNAs. HCT116 cells were transfected with 100ng sense and antisense hnRNPH vectors with H3 or control duplex RNAs. The Dual-Luciferase^® assay was used to evaluate luciferase expression 24 hours post-transfection. In a separate EGFP RNAi experiment, the Steady-Glo^® Luciferase Assay System was used to monitor firefly luciferase activity to normalize transfection of HEK 293 cells. A further RNAi experiment targeted the firefly luciferase gene in the pGL3-Control Vector cotransfected with 20, 2 or 0.4 nM siRNA duplexes into HeLa cells. After 48 hours, the cells were lysed and 10µl tested using the Luciferase Assay System. To test the level of expression of human La antigen targeted for gene silencing, total RNA was harvested from HeLa cells using the SV 96 Total RNA Isolation System, reverse transcribed and used in real-time PCR. (3289)

Notes: In vitro cultures of P. alba were incubated in growth chambers under various conditions. The axenic plants of P. alba were sprayed with either methyl jasmonate (100 lM), methyl salicylate (1mM), or ethanol as a control, and then grown under illumination at 18 hours/day for 24 hours at 25°C. Total RNA was subjected to semi-quantitative RT-PCR using GoTaq^® DNA polymerase and primers for hybrid poplar isoprene synthase. For normalization, actin was used as an external standard. (3355)

Notes: To examine the role of Rac3 in nervous system development, knockout mice were created. Total RNA from postnatal day 0 (P0) and day 9 (P9) brains from wild-type (+/+), heterozygous (+/-), and knockout (-/-) mice was reverse transcribed and a ~260bp product amplified by PCR using GoTaq^® DNA Polymerase and primers for either Rac3 or Rac1 (a Rac3 homolog). No amplification product for Rac3 was observed in the knockout mice. (3328)

Notes: GoTaq^® DNA Polymerase was used in an RT-PCR. Aliquots of RNA were transcribed into cDNA and amplified with GoTaq^® DNA Polymerase using primers specific for Rac1. (3351)

Notes: GoTaq^® DNA Polymerase was used in PCR genotyping of Giardia intestinalis. Primers were designed around a 177 bp sequence of the glutamete dehydrogenase gene (gdh). Typing was based on previously reported assemblages of gdh from cats, dogs, cows and monkeys. PCR was performed in a total reaction volume of 25μl using 1.25 units of GoTaq^® DNA Polymerase.
(3364)

Notes: Type 1 angiotensin II receptor-Renilla luciferase (AT1R-Rluc), and β-arrestin1 and 2 GFP  fusion constructs (βarr1-GFP and βarr2-GFP) were created for BRET protein interaction assays. Combinations of AT1R-Rluc and β-arrestin-GFP constructs were transfected into COS-7 cells. The COS-7 cell cultures were then activated with 100nM angiotensin II in the presence of 60μM EnduRen™ Live Cell Substrate, and BRET fluorescence readings were taken at 475 and 515 nm over a 1 hour period. The authors also describe analysis of helix I mutants of β-arrestin1 and β-arrestin2 in similar β-arrestin-GFP construct BRET studies.  Data are displayed as a ratio of fluorescence readings with both constructs compared to fluorescence from the AT1R-Rluc construct alone.  (3256)

Notes: To examine both the efficiency of gene targeting constructs and the ability to knockin or knockout a gene, recombinant associated adenovirus (rAAV) constructs for p53 and CTNNB1 were created. Either HCT116 or DLD1 human colorectal cancer cell lines were infected with the rAAV and selected for resistance to Geneticin^®. After the drug-resistant colonies were grown for 3–4 weeks, the genomic DNA from each clone was isolated using the Wizard^® SV 96 Genomic DNA Purification System and locus-specific integration assessed by PCR. (3556)

Notes: The authors validated the DNA IQ™ System for manual and automated DNA purification from blood, tissue, bone, hair, epithelial cells and mixed stains such as semen. Automated DNA extraction was performed using the Beckman Coulter Biomek® 2000 workstation. Purified DNA (0.5–1.0ng) was then amplified using the PowerPlex® 16 BIO System and a 6% PAGE PLUS™ gel. Automated DNA purification and use of a single-amplification STR system greatly increased sample throughput. (3645)

Notes: The ability of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to upregulate cytochrome P450 CYP1A1 levels in mouse liver was examined. Total mouse liver RNA was reverse transcribed using the Reverse Transcription System, and the resulting CYP1A1 cDNA was quantitated using real-time PCR. CYP1A1 protein levels were quantitated by Western blot using an anti-CYP1A1 antibody, the Anti-Rabbit IgG (Fc), Alkaline Phosphatase Conjugate secondary antibody and the Western Blue^® Stabilized Substrate for Alkaline Phosphatase.
(3443)

Notes: To examine the methylation status of the DPYD promoter, genomic DNA was extracted from an RKO cell line and from peripheral blood mononuclear cells using the Wizard^® SV Genomic DNA Purification System. The isolated DNA was treated by sodium bisulfate modification followed by amplification and sequencing, or by DHPLC to determine CpG island methylation state. (3587)

Notes: The investigators cloned three β-defensins, which are antimicrobial peptides, from canine testes. A canine expressed sequence tag (EST) was identified based on similarity to human β-defensins. Full-length cDNAs were obtained using 5´- and 3´RACE, then amplified by PCR and cloned into the pGEM^®-T Easy Vector. cDNA sequences were confirmed using the SP6 and T7 Promoter Primers. The tissue-specific expression of canine β-defensins (cBDs) was characterized using the AccessQuick™ RT-PCR System to amplify β-defensin RNA from a variety of tissues. RNA was treated with RQ1 RNase-Free DNase prior to RT-PCR. The identity of the RT-PCR products was confirmed by electrophoresis, transfer to nylon membranes and hybridization to probes derived from sequence-confirmed β-defensin clones; the probes were synthesized using the Prime-a-Gene^® Labeling System. To localize expression of the three β-defensin isoforms in canine testes, in situ hybridization (ISH) was performed. The 3´-RACE products were cloned into the pGEM^®-T Vector, which was then linearized and treated with exonuclease III to delete an approximately 80-bp region shared by the three cBD isoforms. The resulting product was used to synthesize sense and antisense ISH probes. (3453)

Notes: Researchers tested several snail samples from various species for the presence of microsporidia infection. PCR tests were performed with GoTaq^® DNA Polymerase and primers specific to microsporidia 16S DNA sequences. Isolated snail/microsporidia DNA was amplified in 25µl reactions with 0.625 units of GoTaq^® DNA Polymerase. (3378)