Notes: The authors of this study investigated the function of the daf-15 gene in C. elegans. The daf-15 gene encodes the C. elegans ortholog of raptor, a protein involved in the TOR signaling pathway. In C. elegans, daf-15 transcription is regulated by DAF-16, a FOXO transcription factor which is itself regulated by the insulin/IGF-1 signaling pathway. This work links regulation of the TOR pathway, which controls cell growth, to the insulin/IGF-1 pathway, known to affect lifespan, development and metabolism. Three candidate daf-15 genes were amplified by PCR and cloned into the pGEM^®-T Vector. The Riboprobe^® SP6/T7 transcription system was used to transcribe RNA from the candidate clones. Semiquantitative RT-PCR was performed to compare daf-15 mRNA levels in daf-2 and daf-16; daf-2 mutant animals. The mRNA was purified from total worm RNA using the PolyATtract^® mRNA Isolation System. (3633)

Notes: Total RNA was isolated from immature barley anthers with the RNAgents^® Total RNA Isolation System. Total RNA concentration was determined by spectrophotometric analysis. Next, the authors used the PolyATtract^® mRNA Isolation System to isolate poly (A)+ RNA from the total RNA samples. The purified poly (A)+ RNA was used as a template in RT-PCR using the Access RT-PCR System to analyze expression of BEN1 and Bnuc1. (2847)

Notes: Total RNA was isolated from human corneal tissue with the RNAgents^® Total RNA Isolation System.  The total RNA was further processed into the poly(A)+ fraction through the use of the PolyATtract^® mRNA Isolation System. The isolated mRNA was used for RT-PCR and Northern analysis. RT-PCR was performed using M-MLV Reverse Transcriptase. (2569)

Notes: Total RNA was isolated from wildtype and transgenic mouse kidneys and small intestines using the RNAgents^® Total RNA Isolation System. Poly(A)+ RNA was isolated from the total RNA using the PolyATtract^® mRNA Isolation System. Both poly(A)+ and total RNA was used in Nothern blotting. (2578)

Notes: Total RNA was isolated from Arabidopsis tissues using the RNAgents^® Total RNA Isolation System. The RNA was further processed into the poly(A) fraction using the PolyATtract^® mRNA Isolation System. The isolated RNA was used in Northern blot analysis. Blots were probed with a ³²P-labeled RNA probe generated from a cDNA cloned into the pGEM^®-T Easy Vector using the T7 Riboprobe^® in vitro Transcription System. Bioluminescence from transgenic plants containing a firefly luciferase reporter was visualized by spraying the plants with a solution of 5mM Beetle Luciferin, Potassium Salt in 0.1% Triton^® X-100. (2570)

Notes: PolyA+ RNA was isolated from  total RNA from infected or mock-infected chicken embryo fibroblasts using the PolyATtract^® mRNA Isolation System. The isolated mRNA was used to produce ³²P-labeled probes for microarray analysis. The isolated polyA+ RNA was also used to confirm differential expression by Northern blotting. (3205)

Notes: PolyA+ RNA was isolated from mouse 2r15 embryo fibroblast cell line total RNA using the PolyATtract^® mRNA Isolation System. The isolated mRNA was used for Northern analysis and as input mRNA for analysis with an Affymetrix mouse expression gene array. (3204)

Notes: Poly A+ RNA was isolated from the total RNA of three different strains of Saccharomyces cerevisiae, wild-type (JC103), CS165 and JC408, using the PolyATtract^® mRNA Isolation System. The isolated polyA+ RNA was used to make biotinylated cRNAs via a referenced method using AMV Reverse Transcriptase. The resulting probes were used in microarray analysis. (2696)

Notes: The interleukin (IL)-1ß stimulated release of granulocyte macrophage colony stimulating factor (GM-CSF) from lung epithelial cells was explored in this study. To test the promoter activity of GM-CSF, the promoter and enhancer regions were amplified by PCR from human peripheral blood mononuclear cells and cloned into the pGEM^®-T vector. After verification by sequencing, the promoter and enhancer were cloned individually and together into the pGL3-Basic Vector. Additionally, an Xho I/Sal I fragment containing the HSV tk promoter, a gene conferring neomycin resistance, and a poly-A tail were cloned into the Sal I site of pGL3 to allow the production of stable transfectants. To perform stable transfections, 20µl of the Tfx™-50 transfection reagent was incubated with 8µg of plasmid in serum-free medium for 15 minutes at room temperature.  Preconfluent human A549 type II alveolar carcinoma cells were incubated with the transfection mix for 2 hours after washing with serum-free medium. The cells were then cultured in fresh medium for 16 hours before the addition of  0.5mg/mL G-418. After 21 days, foci of cells developed and were harvested for use in luciferase assays. The cells were plated into 24-well plates, grown to confluency and incubated in serum-free medium. Stimulation by 1ng/ml IL-1ß or 1µM PMA for 12 hours was followed by lysate production and measurement of luciferase activity using the Luciferase Assay System and a Turner luminometer. Luciferase readings were standardized against total protein measurements. The PolyATract^® System IV was also used prior to a Northern Blot to obtain purified mRNA. (2742)

Notes: Polyadenylated RNA [poly(A)-RNA] was purified from 0.5 mg total RNA using the PolyATtract® mRNA Isolation System. For primer extension, a primer was radiolabeled by 5' phosphorylation with [γ-³²P]ATP using the Primer Extension System. The primer extension reaction was performed using 10 µg of total RNA from adult Canton-S flies and 100 fmol of a ³²P-labeled primer. Size markers were a ³²P-labeled Promega phiX174/Hinf I Marker standard. (0588)

Notes: Poly(A)⁺ RNA was purified from total RNA by the PolyATtract^® mRNA Isolation System. PCR products were purified by agarose gel electrophoresis and were cloned into pGEM^®-T and pGEM^®-T Easy Vector. Genes were obtained by RT-PCR using the Access RT-PCR System. (1097)

Notes: Poly A(+) RNA was isolated from Rhodospridium toruloides total RNA with the PolyATtract^® mRNA Isolation System. The isolated mRNA was converted to double-stranded cDNA with the Universal Riboclone^® cDNA Synthesis System. (0440)

Notes: Taq DNA Polymerase was used extensively to clone the BLINaC cDNA. The resulting clone was put into the pCI Mammalian Expression Vector. Total RNA was isolated from rat liver and primary hepatocytes with the SV Total RNA Isolation System. The RNA was used for RT-PCR. Northern blotting was performed with poly-A(+) RNA isolated with the PolyATtract^® mRNA Isolation System. (0472)

Notes: The RNAgents^® Total RNA Isolation System was used to isolate total RNA from immortalized human marrow stromal cell lines, HS-5 and HS-27a, that were incubated with 2 ng/mL IL-1β or 5% conditioned media from HS-5 cell cultures. Poly (A)+ RNA was then isolated from total RNA with the PolyATtract^® mRNA Isolation System. The Poly (A)+ RNA was used in RT-PCR  to create amplimers containing T7 promoters. The PCR products were used as templates in in vitro transcription reactions to generate biotin-labeled probes for microarrays. (2760)

Notes: IEC-6 cells were seeded into 96-well plates and cultured in the presence of various concentrations of human IL-17. Cell proliferation was measured using the CellTiter 96^® AQ_ueous Non-Radioactive Cell Proliferation Assay. The pGL3 vector was used to clone a PCR-amplified piece of the 5' flanking region of the CINC gene, and the Luciferase Assay System was used to assess promoter activation. All transfections of IEC-6 cells used the pSV-β-galactosidase vector as a control for transfection efficiency. Total RNA for Northern blotting was isolated using the Poly ATtract System. (2510)

Notes: Poly(A)+ RNA was isolated from total RNA with the PolyATtract® mRNA Isolation System and used for Northern blotting. (0738)

Notes: Poly(A)+ RNA of Drosophila was purified by the PolyATtract^® mRNA Isolation System. (0886)

Notes: This paper describes use of the PolyATtract^® mRNA Isolation System, pCI Mammalian Expression Vector, and TNT^®  Coupled Wheat Germ Extract System for RNA isolation, gene expression and gel-shifts assays (EMSA) respectively. (1477)

Notes: Poly A+ RNA was isolated from Arabidopsis total RNA using the PolyATtract^® mRNA Isolation System. The isolated RNA was used for Northern analysis and both 3' and 5' RACE. The products of the RACE reactions were subcloned into the pGEM^®-T Vector. (1189)

Notes: Total RNA was reverse-transcribed with random hexamer primers and AMV Reverse Transcriptase and then amplified for sequence analysis by nested PCR. Poly(A)+ RNA was purified with the PolyATtract^® mRNA Isolation System. (0382)

Notes: The authors used the PolyATtract^® mRNA Isolation System IV to isolate mRNA from total RNA and the Wizard^® PCR Preps DNA Purification Resin to purify a first-strand cDNA synthesis product.  TNT^® T7 Coupled in vitro Transcription/Translation Reticulocyte Lysate System was used to synthesize radiolabeled DNaseY. And to test for signal peptide function they added Canine Pancreatic Microsomal Membranes (CMMs). (0779)

Notes: The authors isolated mRNA from Drosophila using the PolyATtract^® mRNA Isolation System IV. (1424)

Notes: Poly(A)+ RNA was purified with PolyATtract^® mRNA Isolation Systems. Anti-Rabbit IgG AP Conjugate, and BCIP/NBT were used in Western blotting. R transcripts were synthesized from the Lc cDNA in the pGEM-7Z(+) Vector, and were used in in vitro translation by Rabbit Reticulocyte Lysate Systems to be used as a positive control in Western. (0781)

Notes: Streptavidin MagneSphere^® Paramagnetic Particles (SA-PMPs) were used to capture biotinylated primer probe in a random access retrieval of genetic information by PCR (rargip) screening [ABE, K., (1992) Rapid isolation of desired sequences from lone linker PCR amplified cDNA mixtures: application to identification and recovery of expressed sequences in cloned genomic DNA. Mamm. Genome 2:252-259]. The pGEM^®-T Vector System and PolyATract^® mRNA Isolation System were also used. (0513)

Notes: Poly-A+ RNA was isolated from rat cerebellar total RNA with the PolyATtract^® mRNA Isolation System and used for semi-quantitative RT-PCR. The targets of the RT-PCR were cloned with the pGEM^®-T Vector System and sequenced to show they quantitated the correct targets. (0671)