Notes: This study examined the effect of lactic acid bacteria on gene expression of Staphylococcus aureus in mixed cultures. Total RNA was isolated from a mixed culture of S. aureus and Lactococcus lactis, labeled with 5µg of RNA with Cy^®3- or Cy^®5-dCTP using the ChipShot™ Labeling and Cleanup System, the cDNA was dried and stored at –20°C. Genomic DNA was isolated from S. aureus and L. lactis cultures, digested with HinPII, purified, and 400ng of the digested gDNA labeled with Cy^®3- or Cy^®5-dCTP and the Prime-a-Gene^® Labeling System. The level of gene expression was assessed using a S. aureus microarray. (4068)

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)

Notes: RT-PCR was performed to map the subtypes of nicotinic acetylcholine receptors on A549 cells. cDNA was synthesized using the Reverse Transcription System. Northern blotting to assess XIAP and survivin expression was performed, and probes were labeled using the Prime-A-Gene® Labeling System. Apoptosis was assessed in nicotine-stimulated cells using a DeadEnd™ TUNEL Assay. (3382)

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 used GoTaq^® DNA polymerase to amplify 139bp and 815bp regions of hOST-PTP cDNA for detection and probe synthesis. The full-length 4006bp cDNA was amplified with Pfu DNA Polymerase. Fragments were gel purified using the Wizard^® SV Gel and PCR Clean-Up System prior to cloning into the pGEM^®-T Easy Vector.  The Prime-a-Gene^® Labeling System was used to make ³²P-dCTP labeled probes, which were used to screen cDNA clones. (3111)

Notes: These authors describe a new, quick method of isolating RNA from Streptomycetes coelicolor using five-day-old standing liquid cultures. They report higher yield, better quality RNA and increased purity compared to other methods. S. coelicolor spores were pregerminated, inoculated in liquid media and incubated at 30°C for five days. After incubation, the biomass from six 10 × 10 cm dishes was harvested by scraping the bottom of the dishes and filtering onto a membrane. Then the Mycelium was scraped from the membrane, transferred to a 1.5 ml vial, frozen in liquid nitrogen, and crushed by mortar and pestle. The resulting powder was then transferred to a tube containing 1ml Trizol, processed to extract RNA and the aqueous phase transferred to a new tube for DNase I treatment. Then 375µl of SV RNA dilution buffer was added to the nucleic acid solution, mixed and centrifuged. The resulting supernatant was mixed with 250µl cold 96% ethanol, loaded on an SV column and centrifuged for one minute. The column was then processed as described in the SV Total RNA Isolation System protocol. RNA was eluted from the column by adding 50µl of nuclease-free water and the concentration determined by spectrophotometry. To check the RNA quality, 5µg of the isolated RNA was loaded on a denaturing formaldehyde agarose gel and subjected to electrophoresis for 1 hour or used for Northern hybridizations with a nylon membrane. The 16S DNA probe was labeled with ³²P-dCTP using the Prime-a-Gene® Labeling System. (3070)

Notes: The authors identified a TERMINAL FLOWER homolog, CsTFL, in Washington navel oranges (Citrus sinensis) and investigated its role and the role of other genes in juvenility and flower production. The CsTFL gene was amplified from genomic DNA using PCR and degenerate primers, and amplification products were cloned into the pGEM^®-T Easy Vector. The resulting clones were sequenced using the fmol^® DNA Cycle Sequencing System. The CsTFL cDNA was amplified by RT-PCR, using 4 µg of total RNA from whole flowers and ImProm-II™ Reverse Transcriptase. The amplified cDNA was then cloned into the pGEM^®-T Easy Vector. To evaluate CsTFL gene copy number and allele origins, the authors performed a Southern blot with 10 µg of genomic DNA and a CsTFL probe labeled with the Prime-a-Gene^® Labeling System. To characterize CsTFL expression in various citrus tissues, RT-PCR was performed with 2 µg of total RNA and ImProm-II™ Reverse Transcriptase. The levels of CsTFL RNA and other RNAs were determined by cDNA synthesis using ImProm-II™ Reverse Transcriptase, followed by real-time PCR. Amplification products were quantitated by SYBR^® Green fluorescence. Standard curves for each real-time PCR target were generated using known amounts of in vitro transcribed RNA. Prior to reverse transcription and real-time PCR, total RNA samples were treated with RQ1 RNase-Free DNase to remove DNA contaminants. (3650)

Notes: Rats were fed a 8.0% NaCl diet; aorta explants were taken from these rats and levels TGFβ1 were measured using Promega's TGFβ1 E_max® ImmunoAssay System. Both total and active TGF beta1 levels increased. The Prime-a-Gene® Labeling System was used to make radiolabeled DNA probes. (2340)

Notes: The complete cDNA for the CD1.1 gene was amplified and subcloned into the pTARGET™ Mammalian Expression Vector. The 1020bp, 339 amino acid protein was stably expressed in CHO cells following selection with G-418 sulfate. Expression was confirmed by Northern blot and FACS analysis with an mAb to the CD1.1 protein. The pGEM®-T Vector System was used for routine cloning of both PCR and RT-PCR products. The Prime-a-gene® Labeling System was used create probes for cosmid library screening. (1303)

Notes: The RNAgents^® Total RNA Isolation System was used to isolated total RNA from Porphyromonas gingivalis cultures. The isolated RNA was used for RT-PCR. The Prime-a-Gene^® Labeling System was used to make probes for Southern analysis. (0366)

Notes: The MTS-based CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay was used to quantify the anti-proliferative properties of a Raf-1 antisense oligonucleotide. Human coronary artery smooth muscle cells were grown to 80% confluency in 96 well plates and growth-arrested with medium containing no serum or growth factors. The cells were transfected with the antisense oligonucleotides and then media containing 5% serum added. Cells were assayed for various times (24-96 hours) and with various concentrations of antisense oligonucleotide. Promega’s Prime-A-Gene^® Labeling System also was used in this publication. (1707)