Notes: These authors tested various cell lines for propagation of Zika virus in vitro. Having identified the best cell lines, they performed dose-response experiments with known antiviral compounds, using CellTiter-Glo® to assess cytotoxicity and confirm that candidate compounds were effective in blocking viral replication while having minimal effect on the host cells. The authors also used ImProm-II reverse transcriptase™ and RNasin® ribonulease inhibitor in qRT-PCR assays performed as an alternative to plaque assays for quantifying viral titer. (4940)

Notes: RNasin® Recombinant Ribonuclease Inhibitor was used to protect the in-vitro transcribed FIV R-U5-MA RNA used in the in vitro assembly of FIV Gag Proteins. (4770)

Notes: For RQ-PCR experiments, the authors synthesized cDNA from RNA isolated from TCRγδ+ T cells and frozen RNA in the presence of RNasin® Recombinant Ribonuclease Inhibitor.  (4767)

Notes: PC12 cells were plated and deprived of oxygen and glucose for 5-7 hours. Parallel samples were assessed for viability with the CellTiter-Glo® Cell Viability Assay and cytotoxicity with CellTox™ Green Cytotoxicity Assay (end-point method).  Deprived cells were also analyzed by RT-PCR following RNA extraction with the SV Total RNA Isolation System and converted to cDNA with the ImProm-II™ Reverse Transcriptase in the presence of RNasin® Ribonuclease Inhibitor and PCR performed with the PCR Master Mix for a limited number of cycles.  (4647)

Notes: Polyglutamine diseases are neurodegenerative disorders associated with the presence of proteins containing polyglutamine repeats. These authors studied the mechanism of polygluatmine toxicity. Mutant RNAs carrying an expanded CAG repeat were shown to activate the nucleolar stress pathway and induce apoptosis. Expanded CAG RNAs were shown to interact with nucleolin, preventing it from binding to an upstream control element of the rRNA promoter and causing decreased rRNA transcription, which in turn induced apoptosis. Perturbations in rRNA transcription were identified by real-time PCR, and fluorescence in situ hybridization was used to determine that expanded CAG RNAs localized to the nucleolus. Hybridization solutions were supplemented with RNasin® Ribonuclease Inhibitor. ImProm-II™ Reverse Transcriptase was used in RT-qPCR assays. (4228)

Notes: To better understand the role MOV10 protein, a putative RNA helicase and component of the RNA–induced silencing complex (RISC), in reducing retrotransposon activity, 293T human embryonic kidney cells expressing MOV10 and one of three retrotransposons (LINE1 (L1), Alu or SVA) were lysed in a buffer that included RNasin® Ribonuclease Inhibitor, and FLAG-tagged L1 complexes immunoprecipitated and analyzed by mass spectrometry. Several retrotransposon assays were conducted using FuGENE® HD Transfection Reagent for transfected constructs into 293T, HeLa and HeLa-HA cells. (4255)

Notes: These authors analyzed and compared affinity-purified protein complexes from brain homogenates of wild type and huntingtin (Htt) mutant mice by mass spectrometry. Brain tissue from FLAG-tagged wild-type and Htt mice was homogenized in HEPES buffer supplemented with protease inhibitors and RNasin®. After affinity purification, protein complexes were digested using Sequencing-Grade Modified Trypsin and ProteaseMAX™ Trypsin Enhancer prior to mass-spectrometry analysis. (4227)

Notes: Pentatricopeptide repeat (PPR) proteins are helical repeat proteins that bind specific RNA segments and protect the adjacent RNA by serving as a barrier to exoribonucleases. This study showed that protection by PPR or PPR-like proteins is the predominant mechanism for defining the positions of processed 5′ and intercistronic mRNA termini in land plant chloroplasts. The authors used RNasin^® Ribonuclease Inhibitor in binding reactions between labeled RNA and PPR proteins prior to Gel mobility shift assays. They also used the pGEM^®-T Vector to clone various 3´ RNA terminal sequences amplified by RT-PCR. (4185)

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: RNasin was used in the small RNA library preparation step before sequencing on an Illumina platform.

Notes: These authors investigated the role of the cardiac lineage protein 1 (CLP1/HEXIM1) in skeletal myogenesis. They showed that CLP1 knockout C2C12 cells were unable to differentiate, and then investigated the hypothesis that CLP1 associates with MyoD and HDAC proteins to downregulate cell cycle genes, such as cyclin D1, and allow expression of differentiation-specific genes. RNasin® Ribonuclease inhibitor was used in coimmunoprecipitation assays investigating the interaction between CLP1 and HDAC in C2C12 cells under differentiation and non-differentiation culture conditions. RNasin® was included during cell lysate preparation prior to coimmunoprecipitation assays with antibodies directed against various HDAC proteins. The authors also performed  a luciferase reporter assay using the Dual-Luciferase® Assay System to investigate the regulation of cyclin D1 expression by CLP1, MyoD and HDAC. A luciferase reporter-cyclin D1 construct was co-transfected with MyoD, HDAC and CLP1 constructs and the effect on luciferase expression examined under differentiation and non-differentiation conditions. In differentiation medium MyoD, CLP1 and HDAC5 acted synergistically to reduce cyclin D1 expression. (4225)

Notes: This paper describes a method for detection of polyA RNA in permeablized cells and cell sections. The method is based on incorporation of 5-bromo-2´-deoxyuridine (BrdUTP) into cDNA by AMV reverse transcriptase. The BrdUTP was easily detectable in DNA-RNA duplexes, and undetectable in DNA-DNA duplexes, making it possible to use the method to detect RNA in situ. RNasin® Ribonuclease Inhibitor was used in the reverse transcriptase reaction mix. (4226)

Notes: The authors investigated the role of estrogen inactivation by the SULT1E1-encoded estrogen sulfotransferase in ovulation in mice. Semiquantitative RT-PCR was used to characterize the temporal and tissue-specific expression of SULT1E1 mRNA in ovulating mice. First-strand cDNA synthesis was performed at 37°C for 2 hours using 7.5µg of total RNA, 1µl (0.5µg) of oligo(dT)₁₅, 40 units of RNasin^® Ribonuclease Inhibitor and 200 units of M-MLV Reverse Transcriptase. (3913)

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: Human arsenic methyltransferase (AS3MT) was cloned and mutated to produce allozymes for further analysis. COS-1 cells were transfected with constructs encoding the wildtype enzyme and the synthetic allozymes using TransFast™ Transfection Reagent. The wildtype enzyme and allozymes were transcribed and translated using TNT^® T7 Coupled Reticulocyte Lysate System in the presence of RNasin^® Ribonuclease Inhibitor. (3383)

Notes: Expression levels of transcription factors critical for hemopoiesis in bone marrow were determined using real-time quantitative PCR. First, total RNA was isolated from mouse bone marrow, treated with DNase I, then reverse transcribed using the ImProm-II™ Reverse Transcription System. Each reaction included 2.4µl of 25mM MgCl₂, 4µl of 5X buffer, 1µl of reverse transcriptase, 1µl of dNTP mix (10mM each), 1µl of 50µM random hexamers, 0.5µl of RNasin^® Ribonuclease Inhibitor (20U), and 2µl of sample RNA (200ng/µl). (3454)

Notes: The authors developed a method to reverse transcribe poly(A)+ RNA from leukocytes without using oligo(dT) immobilized on a 96-well plate. Four RNA targets, as well as a synthetic control RNA, were reverse transcribed using MMLV Reverse Transcriptase (1X reverse transcription buffer [50mM KCl, 10mM Tris-HCl (pH 8.3), 5.5mM MgCl₂, 1nL/µL Tween 20], 1.25 mM each dNTP and 4 units of Recombinant RNasin^® Ribonuclease Inhibitor), then quantitated by real-time quantitative PCR. The authors varied the concentration of MMLV Reverse Transcriptase, incubation time, and primer/template ratio] to obtain the maximum yield. Small quantities of MMLV Reverse Transcriptase were sufficient to reverse transcribe short synthetic RNA and abundant RNAs, but approximately 100 units was required for other RNAs. The synthetic control RNA was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. (3456)

Notes: In this study, an in vitro assay was performed to examine the ribonuclease activity of the herpes simplex virus virion host shutoff (vhs) protein. To test whether a purified GST-vhs fusion protein exhibited RNase activity, the entire 3’ UTR of human IEX-1 mRNA (a known target of vhs) was labeled with ³²P and then incubated at 30°C with either GST-vhs or GST alone. Samples were taken at various time points and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. The GST-vhs fusion protein displayed RNase activity. When RNasin^® Plus RNase Inhibitor was added to the reaction, no degradation products were seen, confirming that the nuclease activity of GST-vhs could be blocked by an RNase inhibitor. (3391)

Notes: To prepare single rat NK cells for reverse transcription, the cells were sorted by flow cytometry and lysed using 0.5% NP-40, 2.2µl of 5X M-MLV Reverse Transcriptase Buffer, 0.3µl of 0.1mol/l DTT, 5.5µl DEPC-treated water and 1U of RNasin^® Plus RNase Inhibitor. After a 30-minute incubation on ice, the lysates were heated to 65°C for 90 seconds, cooled to 22°C for 3 minutes and placed back on ice. For the RT reaction, 1U RNasin^® Plus RNase Inhibitor, 60 U of M-MLV Reverse Transcriptase, 2.5mmol/l dNTPs and 100µM primers were added to these single-cell lysates. (3390)

Notes: RNasin^® Plus RNase Inhibitor was added to RNA-DNA ligation reactions and in vitro transcription reactions to protect RNA molecules in both reactions.  For in vitro transcription reactions, 1 unit of RNasin^® Plus RNase Inhibitor was added to the reaction buffer.   (3257)

Notes: MCF-7 cells were incubated for 24 hours in PRF-SFM and then detached and plated on uncoated dishes or dishes coated with 2μg/cm² poly-L-Lysine (P-Lys) in PBS, 30μg/ml fibronectin (Fn) in PBS or 30μg/ml type IV Collagen (Col) in 10mM acetic acid. The cells plated on uncoated dishes were treated both with and without 10nM estradiol (E2). After 24 hours, total cellular RNA was extracted and reverse transcribed using M-MLV reverse transcriptase. Briefly, reverse transcription was performed on 1μg of total RNA in a final volume of 10μl by incubation at 37°C for 30 min with 200U of M-MLV reverse transcriptase, 0.4μg oligo-dT, 0.5μM dNTP and 24U RNasin^® Ribonuclease Inhibitor, followed by heat denaturation for 5 minutes at 95°C. Subsequent PCR analysis was performed on 1μl of the RT product in a final volume of 25μl. Primers were used to amplify the 210bp fragment of PS2 and the 304 bp fragment of cathepsin D. Amplification of 408bp of ribosomal RNA 36B4 was performed as control. The PCR mixture consisted of 1.25U GoTaq^® DNA Polymerase, 1X PCR buffer (10mM Tris-HCl, 50mM KCl), 2.5mM MgCl₂, 0.2mM each dNTP, 0.6μM of each PS2 primer or cathepsin D primer and 0.2μM of each 36B4 primer. PCR was performed for 20 cycles at 95°C for 1 minute, 59°C for 2 minutes and 72°C for 1 minute. Ten microliters of the PCR products were separated on a 1.2% agarose gel. (3377)

Notes: RNA was extracted from 300mg malignant mesothelioma tissue samples and treated with DNase in the presence of RNasin^® Ribonuclease Inhibitor. The RNA was incubated with oligo(dT) primers and reverse transcribed in the presence of radiolabeled nucleotides. The labeled cDNA was hybridized to preprinted microarrays containing 4132 human genes. (2715)

Notes: In this paper, the authors describe a method to label probes for microarrays. The used the RNasin^® Ribonuclease Inhibitor in the probe production process. (2499)

Notes: The patch-clamp technique was combined with RT-PCR in acute hippocampal brain slices. After recording, each cell was harvested to perform transcript analysis and identify subunits that underlie inwardly rectifying K+ currents. The recording pipette solution was supplemented with recombinant RNasin Ribonuclease Inhibitor. After recording channel activity, cell contents were harvested through the recording pipette. RT-PCR was performed on cell contents. (2426)

Notes: The authors describe using the Wizard^® Genomic DNA Purification kit and the SV Total RNA Isolation System to isolate genomic DNA and total RNA, respectively, from Streptococcus pneumoniae. The researchers also added RNasin^® Ribonuclease Inhibitor to purified total RNA before storing it and using it for later RT-PCR reactions performed with the Access RT-PCR System. (2835)