Notes: Here, the downstream targets of c-Myc, a proto-oncogene, are investigated in the context of colorectal cancer (CRC). C-Myc was shown to interact with the promoter region of Deptor through the Gel Shift Assay Systems. Additionally, expression of Deptor was directly correlated with c-Myc/Wnt/β-catenin activation or inhibition. Deptor was also shown to be highly expressed in CRC cells, and downregulation led to differentiation and decreased proliferation. Together, a novel therapeutic target for CRC, Deptor, is presented. (5098)

Notes: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are both neurodegenerative diseases caused by hexanucleotide repeat expansions. The data presented here implicates a model were accumulation of dipeptide repeat proteins (DPR) using non-AUG translation leads to stress induction and disease progression. The Nano-Glo Dual Luciferase assay is utilized with both monocistronic and bicistronic reporters to assess canonical AUG translation and RAN translation. RAN translation of these dipeptide repeat proteins does not require the AUG start codon, 5’-cap or EIF4e. (5127)

Notes: In this study, researchers treated selected viral RNA samples with 1 unit of DNase I to deplete the presence of any genomic DNA in the extracts. The viral RNA genomic regions of interest were reverse transcribed into cDNA using oligo(dT) primer and AMV reverse transcriptase. (4887)

Notes: The authors of this study present a method for genome-wide analysis of miRNA processing intermediates in plants. The method presented uses reverse transcription performed with a mix of primers designed against known miRNA precursors. The molecules are next amplified to generate a library for deep sequencing. Total RNA was isolated and purified from plant tissue and then treated with RQ1 RNase-Free DNase to remove contaminating DNA. Ribosomal RNA was selectively removed before adapter ligation and reverse transcription. cDNA libraries were prepared; PCR products from the libraries were separated on an agarose gel and purified using Wizard® SV Gel and PCR Clean-Up System prior to sequencing. (4561)

Notes: The authors developed a complete method for making cDNA libraries containing miRNA processing-intermediates that used the RQ1 RNase-Free DNase and Wizard® SV Gel and PCR Clean-Up System. (4920)

Notes: The HaloTag^® protein tag was used in experiments to identify protein partners of KDM4A that involved in site-specific copy number gain in tumors, specifically at the 1q12h region. Expression constructs were transfected into HEK293T cells using the FuGENE^® HD Transfection Reagent. The HaloTag-KDM4A (Cat.# FHC00602) and HaloTag-Suv39h1 (Cat.# FHC09879) expression constructs were obtained from the Kazusa DNA Research Institute (Kisarazu, Japan). Interacting proteins identified included DNA polymerase subunits and members of the minichromosome maintenance (MCM) complex. (4408)

Notes: RNA purified from polysome gradient fractions was treated with RQ1 RNase-Free DNase then inactivated by adding 2mM EGTA and heating the reaction to 65°C for 10 minutes. RQ1 RNase-Free DNase was also used to treat total RNA extracted from 3T3–3e5 cells. In both cases, the treated purified RNA was used for cDNA synthesis. (4186)

Notes: The full-length 3´ UTR of mouse RNA-binding protein HuR was amplified and cloned into psiCHECK™-1 Vector to create pEM429 plasmid. To generate radiolabeled RNA substrates for use in cleavage assays, RNA was synthesized from 1µg of linearized plasmid using 50µCi of [α-³²P]UTP, 0.8mM Ribo m⁷G Cap Analog and T7 RNA Polymerase by incubating for 1.5 hours at 37°C. The RNAs were then treated with 1 unit of RQ1 RNase-Free DNase per 1µg of template DNA for 15 minutes at 37°C before extracting with phenol:chloroform, precipitating with ethanol and resuspending in DEPC-treated water. The cleavage assay used 60fmol of ³²P-labeled substrate RNA with 10U Recombinant RNAsin Ribonuclease Inhibitor in a reaction incubated for 2.5 hours at 30°C. RNA probes were labeled with biotin using T7 or T3 RNA Polymerases with a biotin-UTP labeling NTP mixture and incubated for 2 hours at 37°C. The biotinylation reaction was then treated with RQ1 RNase-Free DNase following the same protocol used for radiolabeled RNA. To form HuR/RNA complexes, 2µg of biotinylated RNA was mixed with 100µg nuclear extract and 40 units Recombinant RNAsin^® Ribonuclease Inhibitor in a total volume of 20µl, and incubated for 30 minutes at room temperature. For CstF-64/RNA complexes, 1µg of biotinylated RNA was used and the complexes were stabilized by UV crosslinking using 10U Recombinant RNAsin Ribonuclease Inhibitor during the UV treatment. NIH 3T3 cells were UV crosslinked and either total or nuclear RNA used for immunoprecipitation. After extraction the RNAs were treated with RQ1 RNase-Free DNase for 15 minutes at 37°C before RT-PCR using HuR-specific primers. Total RNA purified from cultured cells were incubated with 50U/ml RQ1 RNase-Free DNase at 37°C for 30 minutes before use in RT-qPCR. (4187)

Notes: The authors investigated the expression of genes encoding histone-like (H-NS) proteins from the self-transmissible pCAR1 plasmid and Pseudomonas putida KT2440 genome, as well as the interaction of H-NS family members in vitro. Gene expression was quantified using quantitative RT-PCR and RNA templates that were treated with RQ1 RNase-Free DNase to degrade contaminating DNA. Interactions between Pmr and other H-NS proteins were monitored using pull-down assays. His-tagged Pmr was expressed in E. coli, purified and used as bait for FLAG-tagged H-NS family proteins. Protein purification of His-tagged proteins was performed using the MagneHis™ Protein Purification System. (4119)

Notes: The authors screened members of a class of acylated hydrazones of salicylaldehydes (INPs) to characterize their ability to inhibit growth of Chlamydia by affecting the type III secretion (T3S) system, a potent virulence mechanism. Expression levels of various T3S genes and gene markers of early, middle and late developmental cycles were examined by RT-PCR in Chlamydia trachomatis-infected HeLa 229 cells in the presence and absence of INPs. HeLa229 RNA was isolated at 4, 8, 24 and 36 hours postinfection, treated with RQ1 RNase-Free DNase and amplified using the Access RT-PCR System in the presence of 0.5 units of RNasin RNase Inhibitor. Each set of experiments included a no-reverse transcriptase control reaction to control for DNA contamination and a positive control reaction using the Positive Control RNA with Carrier and control primers supplied with the kit. (3795)

Notes: The authors examined the locations and levels of apoptosis-related Fas, Fas ligand, Bcl-2, Bax and caspase-3 proteins in ovarian tissue throughout the rat estrus cycle. Protein levels were determined using Western blotting, and proteins were localized by immunohistochemistry. The presence of Fas, Fas ligand, Bcl-2, Bax and caspase-3 mRNA in various ovarian tissues was monitored by RT-PCR. Reverse transcription was performed using the ImProm-II™ Reverse Transcription System, 1µg of RQ1 RNase-free DNase-treated RNA and an oligo(dT) primer. One microliter of the reverse transcription reaction was amplified by PCR, and 10µl of the amplification products were analyzed by agarose gel electrophoresis. (3724)

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: RQ1 RNase-Free DNase was used to remove plasmid DNA template from in vitro transcription reactions.  (3186)

Notes: Two genes (CzcR and CzcS) encoding the CzcCBA (cobalt/zinc/cadmium) heavy metal efflux pump were cloned using  Pfu DNA Polymerase and genomic DNA from PT5 and PT1105 strains of Pseudomonas aeruginosa. The products were 900 and 1,600 bp, respectively. RNA from 5 different strains of Pseudomonas aeruginosa was isolated and treated with RQ1 RNase-Free DNase.  The RNA was used in reverse transcription reactions with the ImProm-II™ Reverse Transcriptase and random primers.  Copy-DNAs produced from the reaction were stored at -20°C until use in real-time PCR.  (3044)

Notes: This paper describes the use of RQ1 RNase-Free DNase to remove DNA from TRIZOL- purified total RNA. The researchers describe stopping RQ1 RNase-Free DNase reactions with RQ1 RNase-Free DNase Stop Solution and heating the reactions at 64°C before using the samples in RT-PCR.  (3187)

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: The Wheat Germ Extract was used to demonstrate that the 3’ end of a viral mRNA acts as a tRNA-like structure (TLS). The tRNA-like structure was shown to accept a [H³]valine residue in the Wheat Germ Extract. The turnip yellow mosaic virus (TYMV) TLS structure was also shown to be able to function with ribosomes by donating [H³]valine in translation assays using Wheat Germ Extract with TYMV RNA but not BMV or AMV RNA. RNAs to be used as templates in the reactions were transcribed with the T7 RiboMAX™ Large Scale RNA Production System. After transcription DNA templates were removed with RQ1 RNase-Free DNase. (3386)

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: Total and poly(A)+ RNA from treated and untreated 8701-BC breast cancer tumor cells were treated with RQ1 RNase-free DNase. The RNA samples were reverse transcribed using the M-MLV RNase H– point mutant reverse transcriptase and random primers to create cDNA used in downstream analyses. The cDNA from the reactions were used in PCR, differential display PCR, and semi-quantitative multiplex PCR reactions. In differential display PCR studies, the authors analyzed differentially displayed bands by staining 6% polyacrylamide gels with the SILVER SEQUENCE™ Staining Reagents. Differentially displayed bands were re-amplified and cloned using the pGEM®-T Easy Vector and high efficiency JM109 competent cells. (3020)

Notes: Total cellular RNA was isolated from peripheral blood leukocytes, mononuclear cells, tumor specimens, and breast cancer cell lines. Purified RNA was treated with RQ1 RNase-Free DNase. (2652)

Notes: Promega's CellTiter 96^® AQ_ueous Non-Radioactive Cell Proliferation Assay was used to examine the effects of adenosine and adenosine receptor agonists on a pituitary folliculostellate cell line and two pituitary endocrine cell lines. The authors also used RQ1 RNase-free DNase during the course of their experiments. (2495)

Notes: Total RNA was isolated from E15, E19, P0, P4 and adult rat brain tissue. RNA was treated with RQ1 RNase-free DNase prior to RT-PCR experiments. (2587)

Notes: In this paper, extracted total RNA was treated with RQ1 RNase-Free DNase prior to RT-PCR analysis. (2365)

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

Notes: A primary cell line from rat subventricular zone which remains undifferentiated over time was developed as a model for MAPK activation. Cell were stimulated with 20 ng/ml bFGF and 20 ng/ml EGF (Promega) to activate MAPK. The MAPK pathway was inhibited with either U0126 (Promega) or  PD98059. Immunocytochemistry was performed with the Anti-ERK 1/2 pAb (1:100 dilution) to detect both active and inactive forms of MAPK proteins and with the Anti-ACTIVE™ MAPK pAb (1:100 dilution) to specifically detect the dually phosphorylated, active forms of MAPK. Cells were also immunostained with the neuron specific marker Anti-III-tubulin mAb (0.5 µg/ml). Cell proliferation was monitored with the CellTiter 96® AQueous One Solution Cell Proliferation Assay System. Apoptosis within the cell population was monitored using the DeadEnd™ Fluorometric TUNEL System. (2391)