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)

Notes: The induction of GADD45α (growth arrest and DNA damage inducible gene 45α) in response to arsenic was examined on the protein and mRNA levels. Protein levels were determined by Western blotting; mRNA levels were determined using the AccessQuick™ RT-PCR System. Changes in GADD45α promoter activity in response to arsenic treatment were monitored in cells transiently transfected with constructs containing GADD45α promoter elements upstream of the firefly luciferase reporter gene. The Dual-Luciferase^® Assay System was used to quantitate luciferase expression, and thus, promoter activity. (3440)

Notes: The host gene E3L is required for vaccinia virus infection and has anti-apoptosis activity. The authors examine the ability of E3L to regulate transcription of several genes related to apoptosis, immune response and viral pathogenesis. IL-6, NF-AT, p53, NF-κB, Ap-1 and cAMP response elements were cloned upstream of a TATA box and the firefly luciferase reporter gene. Renilla luciferase (pRL-null Vector) was used to normalize for transfection efficiency. Luciferase activities were measured using the Dual Luciferase^® Reporter Assay System. The authors also show that the Z-DNA binding region of E3L is important for transcriptional regulation. HeLa cells were transfected with an expression vectors expressing full-length E3L, E3L with a deletion of the Z-DNA binding domain or E3L with point mutations in residues important for Z-DNA binding. The AccessQuick™ RT-PCR System was used to quantitate IL-6, NF-AT and p53 mRNAs; β-actin was used as a control for RNA input. (3452)

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: The tissue-specific patterns of expression of the novel protein disulfide isomerase-like protein of the testis (PDILT) was characterized using the AccessQuick™ RT-PCR System. RNAs isolated from various mouse tissues were amplified to reveal the testis-specific expression. An in vitro PDILT transcript generated using the Riboprobe^® System-T7 was used as a positive control. Actin mRNA was also amplified to demonstrate equal RNA input. (3433)

Notes: The AccessQuick™ RT-PCR System was used to analyze mRNA expression of Hypoxia Inducible Factor-1α (HIF-1α) in HCT116 cells. RT-PCR was performed using 0.5μg of Trizol-isolated total RNA and HIF-1α specific primers. (3060)

Notes: Researchers used the pGEM®-T Vector System to clone the entire 1.4kb Shiga toxin type 2 gene (Stx2) from E. coli O157-H7 C600 (933W). The resultant construct, named pGEMTStx2, was used as a template in PCR to amplify each region of the gene corresponding to Shiga toxin type 2 subunits A and B. Each PCR product was digested with BamHI and EcoRI before ligation into pCDNA 3.1+ (Invitrogen) to create pStx2ΔA and pStx2B. Mice were then immunized with either one or both of these constructs and another construct expressing murine granulocyte-macrophage colony-stimulating factor. Expression of each subunit in mouse tissue was verified by RT-PCR with specific primers and the AccessQuick™ RT-PCR System. (2701)

Notes: The swarming behavior of the symbiotic-pathogenic bacterium Xenorhabdus nematophila is examined in this work. To investigate changes in gene expression in different strains at various times, the researchers utilized the AccessQuick™ RT-PCR System to measure the relative amount of gene product. To ensure that the assay was linear, different cycle numbers were used for each gene target ranging from 15 to 21 cycles of amplification. The RT-PCR used approximately 600ng of total RNA that was treated with RQ1 RNase-Free DNase before use.  (2745)

Notes: The SV Total RNA Isolation System was used to isolate total RNA from HeLa cells for RT-PCR analysis. Fifty nanograms of the total RNA was used as template in AccessQuick™ RT-PCR System reactions. VEGF, ADM, GLUT3, and β-actin mRNA levels in HeLa cells under normoxia and hypoxia conditions were analyzed. (2746)

Notes: Nonobese diabetic (NOD) mice were infected with a nonpathogenic recombinant adeno-associated virus carrying the IL-10 gene. To verify IL-10 transgene expression, total RNA was isolated and IL-10 specific primers were used for amplification using the AccessQuick™ RT-PCR System. (3093)