Notes: To study PHB-mediated gene repression and cell cycle arrest we generated promoter-reporter fusions using the pGL4 Luciferase Vectors. The PHB-E2F1 interaction was investigated using the CheckMate™ Mammalian two-hybrid system. The NanoBiT™ PPI interaction system was used for additional analysis of the PHB-E2F1 interaction. cDNA was synthesized using the Reverse Transcription System. (4900)

Notes: RNA was isolated from adult C. pallens (insect) tissues using the SV Total RNA Isolation System prior to cDNA library construction and sequencing on the Illumina HiSeq™ platform. To validate NGS sequence alignment, end-to-end PCR was performed. PCR products were purified using the Wizard® SV Gel and PCR Clean-Up System prior to cloning into a T vector. To verify the identified differentially expressed genes, quantitative real-time PCR (RT-qPCR) was used. Total RNA was extracted and cDNAs synthesized using the Reverse Transcription System prior to qPCR. (4560)

Notes: Mice RNA samples were converted to cDNA using an oligo-dT primer with the Reverse Transcription System, ethanol precipitated, vacuum dried and transferred to another lab. There they were reconstituted in 20μl of molecular biology grade water.

Detection of caliciviruses in the wild mice samples was attempted using generic calicivirus primers targeting sequences encoding conserved amino acid motifs in the RNA-dependent RNA polymerase (RdRp) region of ORF1. Two microliters of cDNA was used in 25μl PCR reactions using the GoTaq® Green Master Mix. Laboratory mouse RNA samples were tested only with MNV-specific primers in the AccessQuick™ RT-PCR System using 2μl RNA as template.

PCR products were cloned into pGEM-T® Vector and sequenced using M13 forward and reverse primers on an ABI PRISM® 3730 DNA Analyzer. (4330)

Notes: The authors used microarray analysis to identify genes that are differentially expressed in nectaries of Arabidopsis and may be involved in nectar synthesis and secretion. One of these genes was cell wall invertase 4 (cwinv4). The authors generated two Arabidopsis cwinv4 mutant lines to study gene function and used GoTaq® Green Master Mix to confirm the mutant genotype. Expression patterns of cwinv4 in wildtype Arabidopsis and an ortholog from Brassica rapa were examined in various tissues by RT-PCR. The reverse transcription step was performed using the Reverse Transcription System and 0.1µg of RNA. (4093)

Notes: The authors investigated gene transcription within periplasmic flagella of Borrelia burgdorferi, which are composed of a basal body, hook and filament, to determine if hook formation influences flagellin gene expression. They used insertion mutagenesis to construct strains with mutated versions of the hook structural gene flgE that were disrupted by a kanamycin-resistance cassette. The flgE gene and antibiotic-resistance cassette were amplified by PCR and cloned into the pGEM^®-T Vector. To assess the effect of flgE disruption on the transcription of filament proteins FlaA and FlaB, quantitative RT-PCR was performed; enolase was used as an internal control. Negative controls without the reverse transcriptase were included for each sample. (3885)

Notes: The authors investigated the ability of α9β1 integrin to act as a neurotrophin receptor and affect cell signaling pathways. As part of the study, RT-PCR was used to detect the presence of other neurotrophin receptors in their model cell line, SW480. Reverse transcription was performed using the Reverse Transcription System and 1µg of total RNA isolated using the SV Total RNA Isolation System. The resulting cDNA (5µg) was amplified for 35 cycles (β-actin as a control) or 40 cycles (TrkA and p75^NTR) using GoTaq® Green Master Mix. RT-PCR results were confirmed by Western blot analysis. (3884)

Notes: The authors characterized the network of protein-protein interactions for 22 MADS-domain proteins in tomato using yeast two-hybrid and three-hybrid assays. To construct bait and prey proteins, total RNA from various tissues was reverse transcribed using the Reverse Transcription System, then amplified using PCR primers containing restriction enzyme sites for cloning into the bait and prey vectors. (3886)

Notes: The authors investigated the expression pattern of type 1a growth hormone secretagogue receptor (type 1a GHSR), a receptor for ghrelin. In situ RT-PCR was performed on paraformaldehyde-fixed, paraffin-embedded mouse spinal cord tissue. Prior to in situ RT-PCR, tissue sections were treated with proteinase K and triethanolamine, then dewaxed. Reverse transcription was performed using the Reverse Transcription System and oligo (dT)₁₅ primers; followed by amplification using the PCR Master Mix in the presence of 11-digoxygenin-dUTP (1mM). Amplification products were detected using an anti-digoxygenin, alkaline phosphatase-conjugated goat antibody and nitro blue tetrazolium/5-bromo-3-indolylphosphate-p-toluidine salt (NBT/BCIP). (3968)

Notes: The authors examined the regulatory effect of 13-cis-retanoic acid (13-cis-RA) on genes that encode proteins involved in serotonergic neurotransmission in the RN46A-B14 cell line, which was derived from rat embryonic raphe nuclei. Northern blot analysis was performed to quantitate mRNA levels of these genes in 13-cis-RA-treated and untreated cells. cDNA templates for generating Northern blot probes were synthesized by reverse transcription using the Reverse Transcription System followed by PCR. The Reverse Transcription System was also used in RT-PCR to check for the expression of retinoic acid and retinoid X receptors (RAR and RXR, respectively) in RN46A-B14 cells. Briefly, 1µg of total RNA was treated with DNase, reverse transcribed using oligo (dT) primers, then amplified by PCR using RARα, RARβ, RXRα, RXRβ/γ primers. (3790)

Notes: The authors examined the role of ceramide and NF-κB in age-related adipose tissue inflammation in mice. Levels of inflammation-associated molecules, IL-1β, IL-6, TNF-α, COX-2 and peroxisome proliferator-activated receptor (PPAR)-γ mRNA, were quantified by quantitive PCR (qPCR). RNA was isolated from adipose tissues, and first-strand cDNA was synthesized using the Reverse Transcription System prior to qPCR. mRNA levels were also quantified in peritoneal macrophages grown in the presence of young adipocyte-conditioned medium and old adipocyte-conditioned medium. Viability of the primary adipocytes used in these experiments was confirmed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay and CytoTox 96® Non-Radioactive Cytotoxicity Assay. (3777)

Notes: Tumor metastasis suppressor nonmetastatic protein 23 homologue 1 (NM23-H1) interacts with estrogen receptor α (ERα) and influences ERα-mediated gene expression. The authors knocked down NM23-H1 expression using RNA interference in estrogen-treated or untreated MC-7 human breast cancer cells and determined the effect on transcription of estrogen-responsive genes, including progesterone receptor, Bcl-2, cathepsin D and cyclin D1. Levels of these mRNAs were measured in the presence of NM23-H1 or control small interfering RNAs using quantitative RT-PCR. Total RNA was treated with RQ1 RNase-Free DNase to remove contaminating DNA, and cDNA was synthesized using the Reverse Transcription System. The resulting cDNA was subjected to quantitative PCR using SYBR^® Green dye. (3789)

Notes: RT-PCR was performed to map the subtypes of nicotinic acetylcholine receptors on A549 cells. cDNA was synthesized using the Reverse Transcription System. Northern blotting to assess XIAP and survivin expression was performed, and probes were labeled using the Prime-A-Gene® Labeling System. Apoptosis was assessed in nicotine-stimulated cells using a DeadEnd™ TUNEL Assay. (3382)

Notes: Few reliable prognostic markers have been developed for breast cancer. Human tissue kallikreins (hKs) are serine proteases with diverse functions. Fifteen human tissue kallikrein genes (KLK) have been identified. Several human tissue kallikreins have been associated with malignancies, such as hK3 (prostate-specific antigen) and prostate cancer. hK7 has been linked to a significantly poorer prognosis for ovarian and breast cancer, indicating that it may serve as a marker for these diseases. However, previous analyses of KLK7 expression were non-selective studies of all KLK7 mRNA forms. The authors developed a quantitative reverse-transcription-PCR (QPCR) assay, using the Reverse Transcription System on RNA, then developed a highly sensitive QPCR assay, to determine whether a more specific analysis of full-length KLK7 mRNA might lead to similar results as those from previous studies on the multiple KLK7 mRNA forms, with the ultimate goal of determining whether KLK7 could serve as a marker for breast cancer. (3615)

Notes: The ability of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to upregulate cytochrome P450 CYP1A1 levels in mouse liver was examined. Total mouse liver RNA was reverse transcribed using the Reverse Transcription System, and the resulting CYP1A1 cDNA was quantitated using real-time PCR. CYP1A1 protein levels were quantitated by Western blot using an anti-CYP1A1 antibody, the Anti-Rabbit IgG (Fc), Alkaline Phosphatase Conjugate secondary antibody and the Western Blue^® Stabilized Substrate for Alkaline Phosphatase.
(3443)

Notes: Rat liver slices were used to study hepatic stellate cell (HSC) activation and fibrogenesis. The expression of markers of HSC activation and fibrogenesis were analyzed by real-time PCR. To quantitate mRNA levels, total rat liver RNA was reverse transcribed using the Reverse Transcription System, then amplified in the presence of SYBR^® Green. (3430)

Notes: The authors examine the role of brain-derived neurotrophic factor (BDNF) in alcohol addition. RNA was isolated from primary rat hippocampal neurons cultured in the absence or presence of ethanol. The RNA was reverse transcribed using the Reverse Transcription System, and BDNF and GPDH RNAs were quantitated by fluorescent real-time PCR. BDNF and nerve growth factor (NGF) protein levels were monitored using the BNDF and NGF Emax^® ImmunoAssay Systems. (3441)

Notes: These authors characterized cytochrome P450 expression in mouse ovaries treated with the ovotoxin 4-vinylcyclohexene. Total mouse ovary RNA was reverse transcribed using the Reverse Transcription System and random primers, followed by quantitative PCR using primers specific for CYP2E1, 2A and 2B. (3442)

Notes: RNA was extracted from condition-lesioned and unoperated mouse thoracic dorsal root ganglion neurons, and cDNA was generated using Reverse Transcription System. Additionally, RNA was prepared from cut and unoperated mouse sciatic nerve samples using the SV Total RNA Isolation System. (2662)

Notes: NF-E2-related factor-2 (NRF2) upregulates transcription of antioxidant response element (ARE)-driven genes. The authors examined the sensitivity of Nrf-2^–/^– primary mouse neurons to oxidative stress-induced apoptosis using the mitochondrial toxins 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPP⁺) and rotenone. Primary cultures were >090% neurons, as judged by immunostaining with the Anti-βIII Tubulin mAb and other neuron-specific antibodies. Neuronal cytotoxicity in the presence of MPP⁺ and rotenone was monitored using the CellTiter 96^® AQueous Non-Radioactive Cell Proliferation Assay and TUNEL assays. The level of expression of the antioxidant-responsive gene quinone oxidoreductase was measured by mRNA quantitation using RT-PCR, enzyme activity assays and immunocytochemistry. The Reverse Transcription System was used to synthesize cDNA for subsequent amplification by PCR. (3570)

Notes: Reverse transcription and PCR was performed on total RNA isolated from spleens of treated mice to determine the effect of treatment on cytokine mRNA expression using the Reverse Transcription System. (2718)

Notes: Promega reverse transcription buffer, AMV reverse transcriptase, and RNAsin^® RNAse inhibitor were used in reverse transcription reactions of total RNA from HepG2 heptatoma cells. PCR products were cloned into the pGEMT vector. (2613)

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: The Reverse Transcriptase System was used for two step RT-PCR. (0092)

Notes: The pALTER^®-MAX Vector was used to create mutations causing three separate amino acid substitutions on the FKHR cDNA. The Reverse Transcriptase System was used for RT-PCR. (1088)

Notes: Total RNA was isolated from 3-day-old mung bean hypocotyls using the SV Total RNA Isolation System. RT for RT-PCR was performed with the Reverse Transcription System. (0825)