Notes: A novel therapeutic method utilizing synthetic microRNAs combined with a heat shock-inducible promoter to decrease target gene expression is described. This method is ideal for diseases where target gene overexpression leads to disease state. To monitor gene expression, the firefly luciferase gene within a tumor is targeted and expression is monitored using the Dual-Luciferase® Reporter Assay System. The ViviRen™ Live Cell Substrate is used to monitor expression in live mice. (5088)

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: C2C12 myotubes treated with BaF with and without amino acid deprivation were analyzed for gene expression by RT-qPCR. The RNA was isolated with the ReliaPrep™ RNA Cell Miniprep System and converted to cDNA with the ImProm-II™ Reverse Transcription System followed by dye-based qPCR. (4611)

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: Total RNA was isolated using the ReliaPrep™ RNA cell Miniprep System. The Improm-II™ Reverse Transcription System was used to synthesize random hexamer-primed cDNA from 1g of total RNA. (4738)

Notes: 3T3-L1 preadipocytes were treated with various levels of reversine and monitored for induction of apoptosis with the Caspase-Glo® 3/7 Assay with data collected on a GloMax® Instrument. Changes in gene expression of three targets upon reversine treatment were examined through total RNA isolation with the ReliaPrep™ RNA Cell Miniprep System, reverse transcription with the ImProm-II™ Reverse Transcription System followed by dye-based qPCR analysis. (4596)

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: These authors evaluated the effect of knockdown of Parp1 on survival of tumor cells after in vivo delivery of anti-Parp siRNA in a lipid-based nanoparticle format. Inhibition of Parp1 expression in mouse tumors was confirmed by qPCR analysis. Total RNA was extracted from the tumors using TRIzol® reagent (Life Technologies) and reverse transcribed using the ImProm-II™ Reverse Transcription System prior to qPCR using SYBR Green PCR Master Mix (Applied Biosystems) . (4222)

Notes: The authors transfected the myleoid leukemic cell line K562 with the cytosolic sialidase Neu2. Expression of Neu2 resulted in a significant decrease in mRNA levels for the anti-apoptotic factors Bcl-XL and Bcl-2 as determined by real-time PCR. Reverse transcription was carried out with 1µg of total RNA using the ImProm-II™ Reverse Transcription System and random hexamers. cDNA representing 10ng of total RNA was used in a real-time PCR to quantitate Bcl-XL and Bcl-2 mRNA levels. (3725)

Notes: Klotho knockout mice exhibit a phenotype of precocious aging, organ failure, osteoporosis. Humans with specific Klotho alleles are at increased risk for osteoporosis, atherosclerosis and decreased lifespan. The authors of this study looked at the expression of Klotho in CD4+ lymphocytes in patients suffering from rheumatoid arthritis and age-matched healthy individuals. cDNA was prepared from total RNA isolated from purified CD4+ lymphocytes using the ImProm-II™ Reverse Transcription System. Klotho expression, protein level and activity was decreased in the lymphocytes from the RA patients. (3654)

Notes: The authors explored the role of five different eukaryotic initiation factor (eIF) 4GI protein isoforms, which are encoded by alternatively spliced mRNAs, by using short interfering RNAs (siRNAs) to silence the eIF4GI gene. Three eIF4GI siRNA target sequences were evaluated for their ability to reduce eIF4GI mRNA levels in HeLa cells. To quantify the extent of gene silencing, a control plasmid that encodes an eIF4GI/Renilla luciferase fusion mRNA was created using the psiCHECK™-2 Vector. Cotransfection of HeLa cells with the eIF4GI siRNAs and psiCHECK™-2 control plasmid resulted in degradation of the eIF4GI/Renilla luciferase mRNA, leading to reduced Renilla luciferase activity and lower light output. The psiCHECK™-2 Vector encodes the firefly luciferase gene, which allowed normalization of Renilla luciferase expression. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System. Quantitative PCR (qPCR) was used to quantify the silencing of endogenous eIF4GI mRNA splice variants. Prior to qPCR, total RNA was isolated from siRNA-expressing HeLa cells, then reverse transcribed using the ImProm-II™ Reverse Transcription System. qPCR was The pGEM^®-T Easy Vector was used in the creation of plasmids encoding siRNA-resistant eIF4GI isoforms, which were transfected into siRNA-expressing HeLa cells to restore eIF4GI function. (3778)

Notes: Obesity-associated elevated estrogen increases the risk for breast cancer in postmenopausal women. The rate limiting step in the synthesis of estrogen from androgen is catalyzed by the aromatase enzyme. Normally this enzyme is expressed under a weak promoter in adipose tissue; however in breast cancer a second, strong ovary-specific promoter (PII) drives expression of aromatase. This study investigated the relationship of BRCA1 and aromatase expression. RNA isolated from BRCA-1 siRNA-treated adipose stromal cells was reverse transcribed using the ImProm-II™ Reverse Transcription System. The authors show that siRNA knockdown of BRCA1 resulted in activation of the PII promoter, suggesting that BRCA1 can modulate estrogen biosynthesis in adipose tissue. (3606)

Notes: The authors characterize an Arabidopsis cytokinin-receptor mutant. RNA was isolated from leaf tissue from wildtype and transgenic Arabidopsis plants expressing various components of the cytokinin signaling pathway. The RNA was reverse transcribed using the ImProm-II™ Reverse Transcription System, and real-time PCR was performed to quantitate response regulator proteins in the signaling pathway. (3450)

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 immunhistochemistry. 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: 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: Many cancer cells upregulate the co-signaling molecule B7-H1, conferring resistance to anti-tumor immunity. The ability of interferon regulatory factor-1 (IRF-1) to upregulate B7-H1 expression was characterized by cloning fragments of the B7-H1 promoter upstream of the firefly luciferase reporter gene in the pGL3-Basic Vector and monitoring luciferase expression using the Dual Luciferase® Reporter Assay System. Firefly luciferase measurements were normalized using Renilla luciferase (pRL-CMV Vector). Putative IRF-1 binding sites in the B7-H1 promoter were identified using the Gel Shift Assay System. RT-PCR was used to examine B7-H1 mRNA levels in interferon-γ-treated or untreated A549 cells exposed to various concentrations of IRF-1 siRNA. cDNA synthesis was performed with the ImProm-II™ Reverse Transcription System. (3451)

Notes: The expression level of the seven-transmembrane Epstein-Barr virus-induced receptor 2 (EBI2) was measured in peripheral blood mononuclear cells. Total RNA was isolated from T-lymphocytes, B-lymphocytes, monocytes and NK cells, and reverse transcribed using the ImProm-II™ Reverse Transcription System. The resulting cDNA was quantitated using real-time PCR. (3449)

Notes: In this study, GoTaq^® DNA Polymerase was used in two-step RT-PCR. The ImProm-II™ Reverse Transcription System was first used to produce cDNA using an oligo d(T)₁₅ primer. PCR was then performed using GoTaq^® DNA Polymerase. Each reaction contained 2μl cDNA, 10μl GoTaq^® Reaction Buffer, 1μl dNTP (10mM), 0.2μl GoTaq^® DNA Polymerase, 1μl each primer (10pmol) and 34.8μl nuclease-free water. PCR was performed at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 60 seconds for 35 cycles, and 72°C for 10 minutes.PCR products were visualized by agarose gel electrophoresis containing ethidium bromide and then sequenced.
(3368)

Notes: cDNA was synthesized from 5μg total RNA from patient skin fibroblasts using the specific ALG1 β1,4 mannosyltransferase primer in the presence of 5% DMSO and 1 unit of ImProm-II™ Reverse Transcriptase. Reverse transcription reactions were performed at 42°C for one hour.  PCR amplification of the cDNA produced ~1,200bp amplimers.  (3180)

Notes: The authors used real-time quantitative PCR to characterize cytokine response after HIV infection of human lymphoid tissues. To synthesize first-strand cDNA, total RNA was reverse transcribed using the ImProm-II™ Reverse Transcription System: 200U of ImProm-II Reverse Transcriptase, 2µg of total RNA, 500ng of oligo(dT) primer, 500µM dNTPs 3mM MgCl₂ and 24U of RNase inhibitor in 1x ImProm-II™ reaction buffer. To obtain viral stocks for infection, 293T cells were transfected with the proviral plasmids pNL4-3 and pYU-2 using the ProFection^® Mammalian Transfection System—Calcium Phosphate. (3455)

Notes: In this paper,  ImProm-II™ Reverse Transcriptase was used to generate a full length, ~4560 bp cDNA of bovine CD11 messenger RNA from PMA-stimulated BL-3 bovine B cell lymphoma cells. The full length cDNA was amplified using Invitrogen’s Elongase amplification technology. The amplification products were directly sequenced to yield the full sequence of the cDNA. (2848)

Notes: The authors investigate a potential internal ribosome entry site (IRES) in the 5´ untranslated region (UTR) of cellular inhibitor of apoptosis protein 1 (c-IAP1). The c-IAP1 5´ UTR was amplified, cloned into pGEM®-T Vector, sequenced, then inserted into a dicistronic reporter vector between Renilla and firefly luciferase sequences. Using the Dual-Luciferase® Reporter Assay System, IRES activity was evaluated in Rabbit Reticulocyte Lysate and transiently transfected cells. The pSV-β-Galactosidase Control Vector was used as a control for transfection efficiency. Because splicing events were removing part of the Renilla luciferase coding region, the authors chose to use RNA transfection of cells. The ImProm-II™ Reverse Transcription System was used for the reverse transcription step of RT-PCRs to amplify intercistronic regions of the dicistronic RNA to examine mRNA splicing. (3429)

Notes: Improm-II™ Reverse Transcriptase was used to clone U7T snRNA isolated from Drosophila nuclear extracts. The authors used 1ng of extracted U7T snRNA, 30ng of a U7T primer, and 1μl of the Improm-II™ Reverse Transcriptase. cDNAs from the reaction were PCR-amplified and cloned. The resultant clones were used to make probes for Northern blot analysis. (2723)

Notes: ImProm-II™ Reverse Transcriptase was used in real time RT-PCR to measure the ratio of Bax to Bcl-2 in immortalized rat proximal tubular cells (IRPTCs) cultured under normoxic or hypoxic conditions. The researchers used 1μg of total RNA in the reverse transcription reaction. Qiagen’s QuantiTest CYBR Green PCT Kit was used to quantify PCR products. Promega’s terminal deoxynucleotidyl transferase (TdT) was also used for TdT-mediated dUTP nick end labeling (TUNEL) assays on the cells. The TUNEL-stained cells were analyzed by FACS analysis. Data from these experiments was expressed as percent apoptotic cells.  (2849)

Notes: The pGEM^®-T Easy Vector was used to subclone products of a 5´ RACE reaction. A promoter construct, assembled in the pGL3 Basic Vector, was co-transfected with a zebrafish interferon expression vector in the ZF4 zebrafish embryo fibroblast cell line using the TransFast™ Reagent (details provided). Luciferase levels were examined with the BrightGlo™ Luciferase Assay Reagent. Induction of zebrafish mRNA was also examined in zebrafish liver cells (ZFL) following treatment with the known interferon inducer, poly(I)-poly(C). RNA was extracted and reverse transcribed using ImProm-II™ Reverse Transcriptase. The resulting cDNA was used for quantitative, real-time RT-PCR with a SYBR green-based assay. (2627)