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At least 577 records · Page 32Linked to original sources
PubMed2006

Evaluation of NucliSens EasyQ HIV-1 assay for quantification of HIV-1 subtypes prevalent in South-east Asia.

BACKGROUND: Monitoring anti-retroviral therapy requires that viral load assays for human immunodeficiency virus type 1 (HIV-1) be applicable to diverse HIV-1 subtypes. OBJECTIVES: To evaluate NucliSens EasyQ HIV-1 assay for quantitation of common HIV-1 subtypes prevalent in South-east Asia. STUDY DESIGN: One hundred and nineteen plasma samples collected in Hong Kong and Cambodia were used to compare the performance of NucliSens EasyQ HIV-1 and COBAS Amplicor HIV-1 Monitor version 1.5 assays. Viral RNA extracted from the NucliSens MiniMAG was also used for HIV-1 subtyping. RESULTS: Performance of NucliSens EasyQ correlated well with COBAS Amplicor (r=0.777, p<0.001) and the small mean difference (0.0462log(10)IU/mL) obtained in the Bland and Altman model indicated good agreement between two assays. The NucliSens EasyQ assay demonstrated a 95% sensitivity at 500IU/mL and 100% specificity. Reproducibility of this assay was within log(10)2-4IU/mL and had a coefficient of variation between 2.3% and 10.4%. Among the 109 specimens included in the analysis, HIV-1 subtyping identified 64 CRF01_AE, 38 subtype B, 3 subtype C, 3 CRF07_BC and 1 subtype G viruses. CONCLUSIONS: Performance of NucliSens EasyQ was comparable to COBAS Amplicor for HIV-1 viral load monitoring. RNA extracts from NucliSens MiniMAG could be used for HIV-1 viral load monitoring, subtyping and drug resistance mutations detection. Our findings highlight the versatility of both NucliSens EasyQ and COBAS Amplicor in monitoring prevalent subtypes and rare circulating recombinant forms (CRFs) in the South-east Asia region.

Cambodia↗
PubMed1989

Androgen regulation of major mRNAs for proteins of the immature and adult mouse vas deferens.

When total RNA and poly (A)+ RNA extracted from the adult mouse vas deferens were translated in the rabbit reticulocyte lysate protein synthesizing system, they showed a similar electrophoretic pattern of about 20 protein bands of widely varying molecular weights. Within the total mRNA population, three functional mRNAs coding for proteins with molecular weights of 24, 34.5 and 36 kDa showed marked changes after 1 month of castration. The time-course of the response of the translatable proteins showed that while 3 days after castration the 24 and 36 kDa bands were missing, the mRNA coding for the 34.5 kDa band was markedly reduced only 20 days after castration. The negative effect of castration on the three mRNAs could be completely reversed by treatment with testosterone. When total RNA extracted from the immature mouse vas deferens was translated, the 24, 34.5 and 36 kDa protein bands were detectable in 10-day-old males and were synthesized in significant amounts between 10 and 20 days (24 and 36 kDa bands) or between 20 and 30 days (34.5 kDa band). Based on its electrophoretic and immunological properties, the 34.5 kDa protein band, which is predominant in the translation products, was identified as the major androgen-dependent protein previously described in vivo.

Age Factors↗
PubMed1993

Growth hormone-receptor messenger RNA in the rat ovary: regulation and localization.

The results of several reports indicate that GH can modulate ovarian function. In the present study, the expression of the growth hormone-receptor (GH-R) mRNA was studied in the rat ovary using an RNA probe corresponding to a part of the extracellular domain of the GH-R. The probe hybridized to two major transcripts with estimated sizes of 4.0 kb and 1.2 kb in RNA extracted from liver and ovary. Recently, these transcripts have been shown to encode the GH-R and the GH-binding protein (GH-BP). The ontogeny of the GH-R/GH-BP mRNA expression was studied using Northern blot analysis and a solution hybridization RNase protection assay. In the liver GH-R/GH-BP mRNA levels increased with age, while in the ovary, the levels decreased between 1 and 5 weeks of age. Hypophysectomy caused a decrease in GH-R/GH-BP gene expression in the ovary, an effect which could be partly reversed with a single injection of GH (2 mg/kg). No significant changes in the ovarian concentration of GH-R/GH-BP transcripts were seen during the estrus cycle. Using in situ hybridization GH-R/GH-BP transcripts were found to be most abundant in follicles. Northern blot analysis of RNA extracted from isolated granulosa cells and corpora lutea showed that both these compartments contained GH-R and GH-BP mRNA, although more abundant in granulosa cells. Immunoreactive GH-R was detected in granulosa cells of healthy follicles, corpus luteum, and in the germinal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

A sensitive technique to clone low abundance receptor transcripts from single microdissected tissue punches.

Tissue microdissection is a rapidly growing technique with wide applicability in the field of gene expression analysis as improved RNA extraction and reverse-transcription polymerase chain reaction (RT-PCR) techniques provide the sensitivity to amplify transcription products from increasingly small numbers of cells. In spite of these advances, isolation, cloning and regional localization of rare or low-abundance mRNA from very small tissue samples remain a difficult and challenging task, especially when high degenerate primers are to be used. We have addressed this problem using a combination of optimized techniques and purification steps added between individual reaction steps. The extreme sensitivity resulting from these modifications permits cloning of new members of a closely homologous gene family from only one microdissected tissue sample and widens the applicability of tissue microdissection. Using this protocol, nested degenerate PCR primers were designed to amplify members of the large and relatively homologous olfactory receptor (OR) gene family from RNA extracted from 125-microm diameter punches of tissue microdissected from 16-microm sections of the main olfactory bulb (MOB) of the mouse. Levels of OR mRNA in these punches are extremely low, due to the small volume of tissue and the low abundance of OR mRNA in MOB tissue. Several ORs were amplified, cloned and sequenced from a series of individual tissue punches, and in situ hybridization was used to verify the presence of mRNA corresponding to the cloned OR sequences in MOB sections.

Amino Acid Sequence↗
PubMed2004

Effect of formalin on extraction of mRNA from a formalin-fixed sample: a basic investigation.

BACKGROUND: The validity of molecular studies using RNA extracted from decades-old formalin-fixed and embedded tissue blocks is well documented. Formalin-fixed samples are, however, known to be poor materials for molecular biological applications. The aim of this study was to investigate the effect of formalin as a preservation agent on RNA quality and relevant molecular applications. METHODS: The quantity and quality of RNA from liver tissues are modified by the fixation time. Rat liver tissues were harvested and fixed for 4, 8, 12, and 24 h in 10% buffer formalin, and only 9% of RNA could be extracted from the formalin-fixed tissue stored for 4 h. RESULTS: Agarose gel electrophoresis did not show the characteristic pattern of RNA with 18S and 28S in all lanes. Based on the results, it is thought that the RNA was damaged owing to one or more of the following reasons: 1) formalin directly degrades RNA, 2) formalin inactivates RNase, and/or 3) formalin destroys the reagent for extraction of RNA. In this study, it was shown by electrophoresis that very little RNA was extracted by Trizol with 1% formalin. CONCLUSIONS: Formalin has direct and indirect ill effects on RNA during the extraction process.

Animals↗
PubMed2005

Easy and rapid method for the determination of gene expression in cumulus cells incubated for oocyte maturation.

BACKGROUND: The objectives of this study were to develop an easy and rapid method for measuring gene expression in a small number of cells by real-time PCR without RNA extraction and purification, and to use this method to determine more precisely IGF-I gene expression in the cumulus cells surrounding oocytes. METHODS: First, after small numbers of cumulus cells were lysed in cell lysis buffer, they were digested with various concentrations of DNase I for different periods at 37 degrees C to determine the optimal conditions for digestion of genomic DNA in the lysate. Since nonspecific amplification was liable to occur when the non-purified RT product of the cell lysate was used for real-time PCR with the given primers, the optimal conditions for Mg2+ and annealing temperature were well investigated. Further, to create the same conditions as in the actual sample reaction for measurement by real-time PCR, RT-minus product was added to the reaction mixture of the standard curve, and then the amplification efficiency was assessed. Next, IGF-I gene expression in cumulus cells collected from cumulus oocyte complexes (COCs) every 4 h during maturation was determined using the developed method. RESULTS: The optimal conditions for measuring gene expression using the cell lysate from a small number of cells were as follows: incubation of the cell lysate with 0.16 U/microL DNase I with 10 U/microL for 30 min, an Mg concentration of 1.5 mM for amplification of target gene by real-time PCR using RT-product of the cell lysate. When the RT-minus products added to the reaction mixture for the standard curve, which was prepared for purified 18SrRNA plasmid, the PCR efficiency was similar between the sample and the standard. The IGF-I gene expression in the cumulus cells was elevated up through the first 8 h of the culture and then declined gradually by the end of maturation, with the maximal gene expression (778-fold) seen at 8 h. CONCLUSION: It can be concluded that the method developed here, in which equivalent to cumulus cells collected from 0.03-0.075 COCs were employed per reaction, permits rapid and easy determination of target gene expression in a limited number of cells using real-time PCR without RNA extraction.

Animals↗
PubMed2005

Comparative effects of 2 antioxidants, selenomethionine and epigallocatechin-gallate, on catabolic and anabolic gene expression of articular chondrocytes.

OBJECTIVE: . To determine the effects of selenomethionine (Se-met) and epigallocatechin-gallate (EGCg) on gene expression, activation of mitogen-activating kinases, and DNA binding of nuclear factor-kappaB (NF-kappaB) and apolipoprotein-1 (AP-1) in articular chondrocytes. METHODS: Chondrocytes, cultured in low-oxygen tension, were pretreated with L-selenomethionine or EGCg for 24 h, followed by interleukin 1 (IL-1beta) for 1 h (nuclear and cytoplasmic extracts) or 24 h (RNA extraction). Reverse transcription-polymerase chain reaction was performed to determine mRNA levels of matrix metalloproteinases (MMP-1, -3, -13), aggrecanases (-1, -2), IL-1beta, inducible nitric oxide synthase, cyclooxygenases (-1, -2), type II collagen and aggrecan, and transforming growth factor-beta (TGF-beta1, -2, -3) and their receptors I and II. Activity of mitogen-activating protein kinases (MAPK) was assayed by Western blot and AP-1/NF-kB DNA binding by electrophoretic mobility shift assay. RESULTS: Pretreatment with 0.5 microM Se-met prevented IL-1beta-induced MMP-1 and aggrecanase-1 expression, and reduced the cytokine inhibitory effect on type II collagen, aggrecan core protein, and TGF-beta receptor II (TGF-betaRII) mRNA levels. EGCg was more efficient in modulating the effects of IL-1beta on the genes studied. Whereas EGCg inhibited the IL-1beta-activated MAPK, NF-kappaB, and AP-1, Se-met stimulated that signaling pathway. This could account for the differential effects exerted by these antioxidants on chondrocytes. CONCLUSION: Our data provide insights into the mechanisms whereby ECGg and selenium modulate chondrocyte metabolism. Despite their differential mechanisms of action, the 2 compounds may exert global beneficial effects on articular cartilage.

Animals↗
PubMed2005

cDNA expression profiling reveals elevated gene expression in cell clusters overlying focally disrupted myoepithelial cell layers: implications for breast tumor invasion.

BACKGROUND: Our previous studies revealed that a subset of mammary ductal carcinoma in situ (DCIS) contained focally disrupted myoepithelial (ME) cell layers that were predominantly overlain by estrogen receptor (ER) negative cells, which showed a substantially higher rate of cell proliferation and genetic alterations than adjacent ER positive cells within the same duct. This study attempted to assess whether these cells also had a different expression profile on tumor progression related genes. DESIGN: Consecutive sections were made from frozen tissues of 30 DCIS with focally disrupted ME cell layers and associated ER negative cell clusters. ER negative and adjacent ER positive cells within the same duct were microdissected for RNA extraction and amplification. Amplified RNA was converted to biotin-labeled cDNAs and interrogated with 'Cancer PathwayFinder' arrays. RESULTS: Cells within each or among ER negative clusters were immunohistochemically and morphologically similar, whereas they differed substantially from adjacent cells within the same duct. Of 20-paired informative ER negative and positive cells, 15 genes were differentially expressed. Of which, 11(73.3%) were higher in ER negative, 2 (13.3%) were higher in ER positive, and 2 (13.3%) were equal in these cells (p <0.01). Of 11 up-regulated genes in ER negative cells, 8 indirectly or directly promote proliferation and progression, and 3 promote apoptosis. CONCLUSION: ER negative cell clusters showed a significantly higher expressing frequency of multiple tumor progression related genes than their adjacent ER positive counterparts, suggesting that they are likely to be biologically more aggressive and have a greater potential for invasion.

Breast Neoplasms↗