Notes: Total RNA was isolated from various rat tissues with the SV Total RNA Isolation System. Yields are reported as 10µg from 16 whole cochlea, 8µg from 16 modiola, 10.4µg from 48 cochlear sensorineural epithelial/lateral walls and 50µg from the substantia nigra region of four brains. The isolated RNA was used for RT-PCR in the presence of RNasin^® Ribonuclease Inhibitor. The resulting amplimer was subcloned into the pGEM^®-T Easy Vector and clones were purified with the Wizard^® Plus SV Minipreps System. (2176)

Notes: The Wizard^® Genomic DNA Purification Kit was used to isolate genomic DNA from Mycoplasma hyorhinis. The isolated DNA was used for PCR. (1306)

Notes: Total RNA was isolated from 10⁶ actively growing Jurkat, MAW, OZZ or Thp1 cells with the SV Total RNA Isolation System. The equivalent RNA from 8 x 10⁴ cells was used for RT-PCR with the Access RT-PCR System. (2180)

Notes: The SV RNA Total RNA Isolation System was used to isolate total RNA from rat PC12 cells. The isolated RNA was used for RT-PCR in the Access RT-PCR System. (0083)

Notes: The SV Total RNA Isolation System was used to purify total RNA from 30–60mg of human adenoma tumor tissue. The isolated RNA was used for RT-PCR and a Taqman assay. (2177)

Notes: Several cancer-associated proteins, such as the tumor suppressor p53 and the oncoproteins c-ABL and hdm2, have been shown to shuttle between the nucleus and the cytoplasm. Due to this information and the controversy regarding BRCA1 localization, the authors wanted to determine whether BRCA1 is also capable of nuclear-cytoplasmic shuttling.

To this end, a plasmid encoding untagged BRCA1 was created by subcloning the full-length BRCA1 cDNA into a vector as a NotI/ClaI fragment. To generate pYFP-BRCA1, a DNA fragment encoding the yellow fluorescent protein (YFP) was amplified by polymerase chain reaction (PCR) and inserted in frame at the end of the BRCA1 cDNA in the plasmid, using the unique NotI restriction site.

Human breast cancer cell lines T47D and MCF-7, the human breast epithelial cell line HBL-100, and mouse NIH3T3 fibroblasts were seeded onto glass coverslips and transfected at 50–70% confluency with 0.5–2µg of plasmid DNA using the FuGENE® 6 Transfection Reagent. After 48 hours, cells were fixed and processed for fluorescence microscopy. (4283)

Notes: Total RNA was treated with RQ1 RNase-Free DNase prior to RT-PCR. (0816)

Notes: The Wizard^® Genomic DNA Purification Kit was used to purify genomic DNA from the archaeabacterium, Methanosarcina mazei S-6.  The isolated genomic DNA was used in PCR to create templates for sequencing reactions.  (3106)

Notes: Mouse insulinoma betaTC-3 cells were treated with anti-Fas antibody and apoptosis measured with the Apoptosis Detection System, Fluorescein (DeadEnd™ Fluorometric TUNEL System) using flow cytometry. The study also includes a positive and negative control. (0049)

Notes: Total RNA was isolated from Neiserria meningitidis using the SV Total RNA Isolation System. The relative levels of crgA transcript in crgA mutants and wild type strains was determined by RT-PCR with the Access RT-PCR System. (2309)

Notes: Total RNA was isolated from the CNS, spleen and small intestine of mice with preclinical, acute, remission and relapse EAE with the SV Total RNA Isolation System. The isolated RNA was used to analyze gene expression in each stage using the Reverse Transcriptase System and Taq DNA Polymerase in two step RT-PCR. (2163)

Notes: Reverse transcription reaction was performed in the presence of 25ug/ul Single-Stranded DNA Binding Protein and 1 unit/ul RNasin^® Ribonuclease Inhibitor. PCR product and ³⁵S-methionine were added to the TNT^® Coupled Reticulocyte Lysate System to produce labeled protein in a PTT assay. (1185)

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: The SV Total RNA Isolation System was used to isolate total RNA from 100mg of pea seedling leaves. The isolated RNA was used for Northern blots and RT-PCR. Full transcripts of the Pea enation mosaic virus-1 and Pea enation mosiac virus-2 were produced with T7 RNA Polymerase in vitro and translated in Rabbit Reticulocyte Lysate. (2174)

Notes: The Apoptosis Detection System, Fluorescein was used to detect apoptotic nuclei in PC12 cells treated with either calcium ionophores or hydrogen peroxide. Update: The Apoptosis Detection System, Fluorescein has been renamed to DeadEnd™ Fluorimetric TUNEL System. (0503)

Notes: Chromosomal DNA was isolated from V. cholerae with the Wizard® Genomic DNA Purification Kit prior to PCR.  The PCR products were directly purified with the Wizard® PCR Preps System.  The purified 788bp amplimers were used directly for fluorescent Big Dye sequencing. (2299)

Notes: The SV Total RNA Isolation System was used to isolate total RNA from freshly isolated human CD4+ T cells. The isolated RNA was used for RT-PCR with the Access RT-PCR System. (2183)

Notes: The PolyATtract^® System 1000 was used to isolated polyA+ RNA directly from IMR90 cells infected with retroviruses expressing the oncogenic ras or an empty vector control. The isolated polyA+ RNA was used as template in RT-PCR reactions containing ³³P-αdCTP. The radiolabeled targets were then hybridized to microarrays (Genome Discoveries) for differential analysis of IMR90 fibroblast gene expression with the ras oncogene expressing retrovirus or an empty retrovirus control. (2695)

Notes: Total RNA was isolated from dorsal root ganglia neurons and used for RT-PCR and differential display PCR. The RNA was isolated using the RNAgents^® Total RNA Isolation System. (2568)

Notes: Various Candida sp. were treated with a zymolase solution then genomic DNA was isolated with the Wizard^® Genomic DNA Purification Kit. The isolated DNA was used for RAPD analysis with Promega's Taq DNA Polymerase, 10X Thermophilic Reaction Buffer and Nuclease-Free Water. Further diagnostic procedures used Promega's dNTP's and Promega's PCR Nucleotide Mix. (0074)

Notes: The SV Total RNA Isolation System was used to isolate RNA from transfected NIH3T3 cells. The isolated RNA was used for RT-PCR. (2159)

Notes: The SV Total RNA Isolation System was used to isolate total RNA from rat kidney medulla and medullary thick ascending limb. The isolated RNA was used in a quantitative RT-PCR. (0073)

Notes: The RNAgents^® Total RNA Isolation System was used to isolate total RNA from mouse splenocytes, splenic B cells and embryonic fibroblasts. The isolated RNA was used in Northern blots and semi-quantitative RT-PCR. (2567)

Notes: The DeadEnd™ Colorimetric Apoptosis Detection System (DeadEnd™ Colorimetric TUNEL System) was used to analyze apoptotic nuclei in rat gastric sections in response to aspirin. Formalin-fixed, paraffin embedded 40µm sections were analyzed. Cells with nuclei staining dark brown (TUNEL positive) and showing the morphological symptoms of apoptosis were counted to quantitate the number of apoptotic cells. A lot detail is provided about the counting procedure. (1506)

Notes: The Access RT-PCR System was used to assess whether some uncharacterized E. coli sequences that have putative open reading frames are in fact transcribed. The RT-PCR samples contained 0.1µg of bacterial total RNA, and were amplified for 35 cycles. (2158)