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RNA Isolation (rna + isolation)
Selected AbstractsSerotyping and genotyping of HIV-1 infection in residents of Khayelitsha, Cape Town, South AfricaJOURNAL OF MEDICAL VIROLOGY, Issue 12 2006G.B. Jacobs Abstract It is estimated that between 5.5 and 6.1 million people are infected with HIV/acquired immunodeficiency syndrome (AIDS) in South Africa, with subtype C responsible for the majority of these infections. The Khayelitsha suburb of Cape Town has one of the highest HIV prevalence rates in South Africa. Overcrowding combined with unemployment and crime in parts of the area perpetuates high-risk sexual behavior, which increases exposure to infection by HIV. Against this background, the objective of this study was to characterize HIV-1 in residents confirmed to be seropositive. Serotyping was performed through a competitive enzyme-linked immunosorbent assay (cPEIA). Genotyping methods included RNA isolation followed by RT-PCR and sequencing of the gag p24, env gp41 immunodominant region (IDR), and env gp120 V3 genome regions of HIV-1. With the exception of a possible C/D recombinant strain, all HIV-1 strains were characterized as HIV-1 group M subtype C. One individual was shown to harbor multiple strains of HIV-1 subtype C. In Southern Africa, the focus has been to develop a subtype C candidate vaccine, as this is the major subtype found in this geographical area. Therefore, the spread of HIV-1 and its recombinant strains needs to be monitored closely. J. Med. Virol. 78:1529,1536, 2006. © 2006 Wiley-Liss, Inc. [source] Development and demonstration of RNA isolation and RT,PCR procedures to detect Escherichia coli O157:H7 gene expression on beef carcass surfacesLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2000E.D. Berry Preventing the development of pathogen resistance to processing and preservation techniques will require an understanding of the genetic mechanisms that pathogens use in situ to adapt and develop tolerance to stresses they encounter in the food environment. RNA isolation and reverse-transcription (RT),PCR protocols were developed as tools to detect gene expression in bacteria on beef carcass surfaces. The utility of these procedures was demonstrated by detecting the expression of a selectively-inducible green fluorescent protein (GFP) gene in a plasmid-transformed strain of Escherichia coli O157:H7 inoculated onto beef carcass surface tissue. These procedures should serve as useful tools for studying the genetic responses of bacteria when exposed to antimicrobial interventions applied to food animal carcasses. [source] The expression of androgen-responsive genes is Up-Regulated in the epithelia of benign prostatic hyperplasiaTHE PROSTATE, Issue 16 2009Katherine J. O'Malley Abstract BACKGROUND Benign prostatic hyperplasia (BPH) is one of the most common diseases among aging men in the United States. In addition to aging, the presence of androgens is another major risk factor in BPH development. However, whether androgen signaling is altered in BPH remains unclear. To determine androgen signaling in BPH, we characterized the expression of four different androgen-responsive genes, Eaf2/U19, ELL2, FKBP5, and PSA, in BPH and adjacent normal glandular epithelial cells. METHODS A set of 17 BPH specimens were resected from patients over 60 years of age with clinical symptoms of BPH. Laser-capture microdissection (LCM) was used to isolate glandular epithelial cells from BPH areas and adjacent normal areas, separately. LCM isolated cells from individual specimens were lysed and RNA isolation, reverse transcription, and real-time PCR were performed using CellsDirectÔ One-Step qRT-PCR Kit (Invitrogen, Carlsbad, CA). RESULTS All of the assayed genes displayed increased expression, from ,2- to ,6-fold, in BPH as compared to the adjacent normal epithelial cells. We also generated a composite androgen response index based on the expression levels of the four genes, which provides a reliable readout for overall androgen action. Our study showed that the composite androgen response index in BPH is ,4-fold as compared to that in the adjacent normal tissues. CONCLUSIONS Androgen signaling is significantly elevated in BPH relative to the adjacent normal prostate. Understanding the mechanisms causing elevated androgen signaling may lead to novel approaches for prevention and/or treatment of BPH. Prostate 69: 1716,1723, 2009. © 2009 Wiley-Liss, Inc. [source] Quantitative RT-PCR analysis of estrogen receptor gene expression in laser microdissected prostate cancer tissueTHE PROSTATE, Issue 8 2009Thomas J. Walton Abstract BACKGROUND Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ER,) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. METHODS Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ER,), estrogen receptor alpha (ER,), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann,Whitney U -test. Correlation coefficients were analyzed using Spearman's test. RESULTS Significant positive correlations were seen when AR and AR-dependent PSA, and ER, and ER,-dependent PGR were compared, indicating a representative population of RNA transcripts. ER, gene expression was significantly over-expressed in the cancer group compared with benign controls (P,<,0.01). In contrast, PGR expression was significantly down-regulated in the cancer group (P,<,0.05). There were no significant differences in AR, ER, or PSA expression between the groups. This study represents the first to show an upregulation of ER, gene expression in laser microdissected prostate cancer specimens. CONCLUSIONS In concert with recent studies the findings suggest differential production of ER, splice variants, which may play important roles in the genesis of prostate cancer. Prostate 69: 810,819, 2009. © 2009 Wiley-Liss, Inc. [source] Microarray analysis of changes in renal phenotype in the ethylene glycol rat model of urolithiasis: potential and pitfallsBJU INTERNATIONAL, Issue 4 2004Daniel H.-C. OBJECTIVES To investigate, in an initial study, the use of microarray analysis (MA) to develop an information base for urolithiasis. MA enables the screening of thousands of genes simultaneously making it the technique of choice for situations where the results are known, but the underlying mechanisms are not. Little is known about the pathological changes occurring in the kidney during urolithiasis and this has severely hampered efforts to develop effective therapeutics. MATERIALS AND METHODS Male rats were treated with 0.75% ethylene glycol for 2, 4 or 8 weeks; after death the kidneys were processed for RNA isolation and MA, conducted using a rat-based chip (one kidney/chip) and the results confirmed by reverse transcription-polymerase chain reaction (RT-PCR, 21 probe sets; control, four rats; treated, five rats). Targets were defined as different by the software if the fold change (FC) was ,,2, and sorted into functional categories using a data-mining tool. The repeatability of MA was investigated by subjecting the 4-week samples to MA in two independent runs. RESULTS The results for targets with a FC of , 2 were plotted (y = 1.01x , 0.75; r2 0.84). Comparing the results obtained by RT-PCR and MA showed a good qualitative correlation for those targets having a FC of ,,5 as determined by MA. Changes in the expression of genes associated with tubule function and regulation, oxidative damage, and inflammation were the most common in the functional categories. Changes in the expression of tubule-specific markers indicated that there was damage to the proximal (,-adducin, organic anion and cation transporters, sodium-hydrogen exchange protein-isoform 3) and distal tubules (,-adducin, kallikrein) at 2 and 4 weeks. Increased expression of mitochondrial uncoupling protein indicated that there were changes to the mitochondria and oxidative stress at 2 and 4 weeks. CONCLUSION This study shows the power of MA as an exploratory technique, and changes in the expression of several physiologically important genes whose expression has not previously been reported to be affected by hyperoxaluria or calcium oxalate crystalluria. [source] | |||||||||||||