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At least 703 records · Page 39Linked to original sources
PubMed2003

Differential gene expression in pubococcygeus muscle from patients with pelvic organ prolapse.

OBJECTIVE: This study was undertaken to compare differential gene expression in the pubococcygeus muscle in patients with pelvic organ prolapse and controls. STUDY DESIGN: We performed microarray analysis on individual pubococcygeus muscle biopsy specimens from five patients with stage III or IV pelvic organ prolapse and five control subjects without prolapse. This study received full Institutional Review Board approval. Total RNA was extracted, purified, and probed on the Human Genome U95A Array for each individual sample. RNA from patients and controls was not pooled. For microarray analysis, 7 microg of total RNA was used to synthesize complementary DNA that was then biotinylated. Arrays were hybridized for 16 hours in the GeneChip Fluidics Station 400 and were washed and scanned with the Hewlett-Packard GeneArray Scanner. Affymetrix GeneChip 5.0 software was used for scanning and data analysis. RESULTS: Of the 12626 total genes compared, 257 genes were more than 2-fold underexpressed, 20 genes were more than 5-fold underexpressed, and 3 genes were more than 10-fold underexpressed in patients with pelvic organ prolapse compared with control subjects. Myosin-binding protein H was 24.7 times underexpressed in patients with prolapse (normalized signal intensity [NSI]: 0.46 [0.2-0.6]) compared with controls (NSI: 11.4 [0.2-31.3]). Skeletal muscle myosin heavy polypeptide 3 was 17.4 times underexpressed in patients with prolapse (NSI: 0.85 [0.7-0.9]) compared with controls (NSI: 14.8 [1.5-38.3]). Of the 12,626 genes compared, 479 genes were more than 2-fold overexpressed, 18 genes were more than 5-fold overexpressed, and 2 genes were more than 10-fold overexpressed in patients with pelvic organ prolapse compared with controls. Many of these overexpressed genes were related to actin and myosin proteins. Smooth muscle myosin heavy chain was 11.8 times overexpressed in patients (NSI: 5.21 [0.25-22.71]) compared with controls (NSI 0.44 [0.11-0.71]). Myosin light-chain kinase was 5.8 times overexpressed in patients (NSI: 7.9 [0.5-36.1]) compared with controls (NSI: 1.37 [0.38-1.8]). Extracellular matrix proteins were also differentially regulated. Cartilage oligomeric matrix protein precursor was found to be 6.0 times underexpressed, whereas tenascin-C (hexabrachion) was 5.1 times overexpressed in prolapse patients. CONCLUSION: These data suggest that the differences between patients with advanced pelvic organ prolapse and controls may be related to differential gene expression of structural proteins that are related to actin and myosin as well as extracellular matrix proteins in the pubococcygeus muscle. Studies are ongoing to confirm these findings and to further characterize the role of these genes in prolapse.

Actins↗
PubMed2003

[Cloning and expression of human canstatin and its purification and bioactivity].

Total RNA was extracted from placenta umbilical tissue and the canstatin cDNA was amplified from total RNA by net-RT-PCR technique. The amplified cDNA was cloned into pSP72 and sequenced. The canstatin cDNA was cut down from pSP72C with BamH I / Nde I and ligated into the vector pET-3c. The resultant plasmid pETC was then transformed into E. coli BL21 (DE3). The canstatin gene was efficiently expressed after IPTG induction as a 25 kD band on SDS-PAGE. The expressed product constituted approximately 27.9% of the total bacterial proteins estimated by densitometry and existed mainly as inclusion body. The inclusion bodies were washed, lysed, refolded and purified on the Sephadex G-75 gel filtration column to a purity of 91.4%. CAM assay showed that 10 microg purified canstatin is enough to inhibit the angiogenesis of chichen embryo microcapillary vessel.

Angiogenesis Inhibitors↗
PubMed1996

Respective degree of expression of beta 1-, beta 2- and beta 3-adrenoceptors in human brown and white adipose tissues.

1. The possible existence of a beta 3-adrenoceptor in human brown and white adipose tissues was investigated by mRNA expression and binding studies. 2. The relative amounts of beta 1-, beta 2- and beta 3-adrenoceptor mRNA, as determined by total RNA Northern blot analysis in newborn brown adipose tissue, were 28, 63 and 9% respectively of the total beta-adrenoceptor mRNA. 3. The beta 1/beta 2-adrenoceptors of human brown adipose tissue plasma membranes were characterized using [3H]-CGP 12177 as a ligand. Their Kd and Bmax values were 1.9 nM and 156 fmol mg-1 of membrane proteins, respectively. The beta 3-adrenoceptor was characterized by use of the new specific radioligand [3H]-SB 206606. The binding of this ligand was stereospecifically displaced by the active R,R- or the inactive S,S-enantiomer of BRL 37344 up to a concentration of about 10 microM. The Kd and Bmax values of the brown adipose tissue membrane beta 3-adrenoceptors were 87 nM and 167 fmol mg-1 of proteins, respectively. A low affinity [3H]-CGP 12177 binding site population was also detected in these membranes. 4. In human omental white adipose tissue, no beta 3-adrenoceptor mRNA could be detected in total RNA Northern blots and the beta 1-and beta 2-adrenoceptor mRNAs represented 9 and 91%, respectively of the total beta-adrenoceptor mRNA, and no specific binding of [3H]-SB 206606 could be measured.

Adipose Tissue↗
PubMed1996

Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo.

Immunization with defined tumor antigens is currently limited to a small number of cancers where candidates for tumor rejection antigens have been identified. In this study we investigated whether pulsing dendritic cells (DC) with tumor-derived RNA is an effective way to induce CTL and tumor immunity. DC pulsed with in vitro synthesized chicken ovalbumin (OVA) RNA were more effective than OVA peptide-pulsed DC in stimulating primary, OVA-specific CTL responses in vitro. DC pulsed with unfractionated RNA (total or polyA+) from OVA-expressing tumor cells were as effective as DC pulsed with OVA peptide at stimulating CTL responses. Induction of OVA-specific CTL was abrogated when polyA+ RNA from OVA-expressing cells was treated with an OVA-specific antisense oligodeoxynucleotide and RNase H, showing that sensitization of DC was indeed mediated by OVA RNA. Mice vaccinated with DC pulsed with RNA from OVA-expressing tumor cells were protected against a challenge with OVA-expressing tumor cells. In the poorly immunogenic, highly metastatic, B16/F10.9 tumor model a dramatic reduction in lung metastases was observed in mice vaccinated with DC pulsed with tumor-derived RNA (total or polyA+, but not polyA- RNA). The finding that RNA transcribed in vitro from cDNA cloned in a bacterial plasmid was highly effective in sensitizing DC shows that amplification of the antigenic content from a small number of tumor cells is feasible, thus expanding the potential use of RNA-pulsed DC-based vaccines for patients bearing very small, possibly microscopic, tumors.

Animals↗
PubMed2000

The influence of photoperiodic growth condition on isolation of RNA from strawberry (Fragaria x ananassa Duch.) tissue.

Purification of high-quality RNA from different strawberry tissues is often affected by the presence of high levels of contamination by polysaccharides and phenolic compounds. With the protocol detailed here we describe for the first time total RNA purification from petiole tissue. Treating the plants used as source of material with short-daylight regime prior the extraction we are able to obtain RNA suitable for further applications such as in vitro translation, RT-PCR, and RNA blot analysis. The yield of total RNA extraction is significantly enhanced when tissue from plants grown under short-day photoperiodic condition is used compared with that taken from plants grown under long day photoperiod.

Electrophoresis, Agar Gel↗
PubMed1986

Human type III collagen gene expression is coordinately modulated with the type I collagen genes during fibroblast growth.

Type III collagen is one of the major interstitial collagens and, as such, plays an important role in modulating the structure and function of most tissues. To compare the expression of the type III collagen gene to that of the type I collagen alpha 1(I) and alpha 2(I) genes, cDNAs encoding the 3' one-third of the human alpha 1(III) collagen mRNA were obtained by screening a human fetal lung fibroblast cDNA library with a cloned segment of the chicken alpha 1(III) gene. Northern blot analysis of human fetal lung fibroblast RNA demonstrated two alpha 1(III)-specific mRNAs of sizes 6.6 and 5.8 kilobases, sizes clearly different from those of the type I collagen mRNAs. Analyses of populations of dividing and nondividing human lung fibroblasts revealed that, on a per cell basis, the nondividing population contained twice as much alpha 1(III) mRNA than did the dividing population. The same was true for the type I collagen alpha 1(I) and alpha 2(I) mRNA transcripts. Similar results were obtained when alpha 1(III), alpha 1(I), and alpha 2(I) mRNA transcripts were quantified by using dot blot evaluation of total RNA, Northern analysis of total RNA, and dot blot evaluation of cytoplasmic RNA. Thus, despite the fact that the alpha 1(III) collagen gene is located on a chromosome different from the alpha 1(I) and alpha 2(I) genes, the expression of these three collagen chains appears to be coordinately controlled during periods of rapid and slow fibroblast growth.

Amino Acid Sequence↗
PubMed1986

Thyroglobulin gene activation by thyrotropin and cAMP in hormonally depleted FRTL-5 thyroid cells.

Thyroglobulin gene control by cAMP and thyrotropin has been compared in FRTL-5 thyroid cells maintained in the absence of serum, insulin, cortisol, and thyrotropin for 7 days. Readdition of thyrotropin induces an increase in the expression of the thyroglobulin gene with little effect on total RNA synthesis. In contrast, addition of 8-bromo cyclic AMP and cholera toxin stimulate total RNA synthesis as well as thyroglobulin gene expression. The same non-selective effect of cyclic AMP on the thyroglobulin gene was observed when transcriptional activity was measured in permeabilized FRTL-5 cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2003

Gene expression and intracellular pathways involved in endothelial dysfunction induced by VLDL and oxidised VLDL.

OBJECTIVES: The molecular mechanisms underlying the relationship between elevated plasma concentrations of triglyceride-rich lipoproteins and coronary artery disease remain uncertain. In the present work, we investigated the gene expression pattern and intracellular pathways in human endothelial cells incubated with very low density lipoproteins (VLDL). Moreover, as VLDL can enter the arterial wall and undergo oxidative modification, we compared the VLDL-induced expression pattern with the one of oxidised VLDL (Ox-VLDL). METHODS: Total RNA from endothelial cells incubated with 75 microg/ml VLDL or Ox-VLDL and total RNA from endothelial cells under basal conditions were hybridised to identical microarrays containing 8411 genes. Seven clusters of expression profiles were identified. This pattern was validated by quantitative real-time PCR of selected genes. The intracellular pathway involved in VLDL or Ox-VLDL mediated endothelial responses were also investigated. RESULTS AND CONCLUSION: VLDL predominantly activated the ERK1/2 pathway while P38 MAPK was the main target of Ox-VLDL. CREB and NF-kappa B were activated by both VLDL and Ox-VLDL. Real-time PCR demonstrated that VLDL induced matrix metalloproteinase-2 (5.47+/-1.74 fold), CD38 (2.38+/-0.23) and transforming growth factor-alpha (2.51+/-0.30) expression. Ox-VLDL was found to induce interleukin-15 (2.10+/-0.48) and macrophage migration inhibitory factor (3.19+/-0.07) expression. In addition, several genes implicated in endothelial cell activation and damage/proliferation were identified by the array analysis. Ox-VLDL was found to promote the generation of reactive oxygen species and exert a cytotoxic effect, while VLDL lacks these effects. These findings confirm the involvement of VLDL and Ox-VLDL in endothelial dysfunction and suggest new genes and molecular mechanisms involved in these actions.

Cell Death↗
PubMed2004

Gene-expression profiling using suppression-subtractive hybridization and cDNA microarray in rat mononuclear cells in response to welding-fume exposure.

Welders with radiographic pneumoconiosis abnormalities have shown a gradual clearing of the X-ray identified effects following removal from exposure. In some cases, the pulmonary fibrosis associated with welding fumes appears in a more severe form in welders. Accordingly, for the early detection of welding-fume-exposure-induced pulmonary fibrosis, the gene expression profiles of peripheral mononuclear cells from rats exposed to welding fumes were studied using suppression-subtractive hybridization (SSH) and a cDNA microarray. As such, Sprague-Dawley rats were exposed to a stainless steel arc welding fume for 2 h/day in an inhalation chamber with a 1107.5 +/- 2.6 mg/m3 concentration of total suspended particulate (TSP) for 30 days. Thereafter, the total RNA was extracted from the peripheral blood mononuclear cells, the cDNA synthesized from the total RNA using the SMART PCR cDNA method, and SSH performed to select the welding-fume-exposure-regulated genes. The cDNAs identified by the SSH were then cloned into a plasmid miniprep, sequenced and the sequences analysed using the NCBI BLAST programme. In the SSH cloned cDNA microarray analysis, five genes were found to increase their expression by 1.9-fold or more, including Rgs 14, which plays an important function in cellular signal transduction pathways; meanwhile 36 genes remained the same and 30 genes decreased their expression by more than 59%, including genes associated with the immune response, transcription factors and tyrosine kinases. Among the 5200 genes analysed, 256 genes (5.1%) were found to increase their gene expression, while 742 genes (15%) decreased their gene expression in response to the welding-fume exposure when tested using a commercial 5.0k DNA microarray. Therefore, unlike exposure to other toxic substances, prolonged welding-fume exposure was found to substantially downregulate many genes.

Air Pollutants, Occupational↗
PubMed1999

Aromatase and sex steroid receptors in human vena cava.

Among sex steroids, especially estrogen metabolism has been considered to play a role in the function and pathology of human veins. We investigated the expression and activity of the estrogen-producing enzyme aromatase and estrogen receptor (ER) in human vena cava to assess possible in situ biosynthesis of estrogens and their modes of action. We first examined aromatase expression by immunohistochemistry in human inferior vena cava obtained from 29 autopsy cases (11 males, 18 females, 63.6 +/- 3.0 years old). We then semiquantitated the level of aromatase mRNA by reverse transcriptase-polymerase chain reaction in 24 cases and aromatase activity by 3H-water assay in 15 cases to examine whether or not and in which cell types aromatase was expressed. We also studied alternative use of multiple exon 1s of its gene and immunolocalization of 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD I), which converts estrone produced by aromatase to estradiol, a biologically active estrogen and ER. Aromatase and 17beta-HSD I immunoreactivity were both detected in smooth muscle cells (SMC) of the media in all the cases and in endothelial cells (EC) in 20 and 22 cases, respectively. ER immunoreactivity was detected in SMC of vena cava in 21 cases. The amount of aromatase mRNA was significantly greater in the cases utilizing 1c (I.3) or 1d (P.II) of exon 1 (9 cases, 191.1 +/- 26.3 attomol/ng total RNA) than those utilizing 1b (I.4) as the promoter (14 cases, 50.6 +/- 13.0 attomol/ng total RNA) (p < 0.01). Significant correlation (p < 0.05) was observed between the amount of aromatase mRNA and aromatase activity in 15 cases examined. No significant correlation was detected between the amount of aromatase mRNA or aromatase labeling index and the ER status. These results suggest that estrone and estradiol are produced in the human vena cava and that their production is mediated by aromatase and 17beta-HSD I, respectively but not all of these locally synthesized estrogens may not work directly in situ.

17-Hydroxysteroid Dehydrogenases↗
PubMed2003

[Expression of urotensin II mRNA in normal adrenal medulla and pheochromocytoma tissues].

OBJECTIVE: To investigate the expression of human urotensin II (UII) mRNA in normal adrenal medulla and pheochromocytoma tissues. METHODS: The total RNA of normal adrenal medulla from 9 males who died of accidents and the total RNA of pheochromocytoma tissues resected from 19 patients were extracted. The expression of UII mRNA was studied by reverse transcription-polymerase chain reaction (RT-PCR). UII and its internal control GAPDH were co-amplified in the same tube. Semi-quantitative analysis of the mRNA expression level of UII was carried out based on the ratios of the grey scale of the amplified target band to that of the control band (UII/GAPDH). RESULTS: The amplified product of UII is a 385 bp fragment and 99.7% of the bases were the same as the sequence registered in the GenBank. The UII/GAPDH in the normal adrenal medullae and pheochromocytomas were 0.69 +/- 0.29 (n = 9) and 0.22 +/- 0.18 (n = 19) respectively (P < 0.05). CONCLUSION: Both the normal adrenal medulla and pheochromocytoma express UII mRNA. The expression of UII mRNA is lower in pheochromocytoma than in normal adrenal medulla.

Adrenal Gland Neoplasms↗
PubMed1984

Expression of pregnancy-specific genes in preneoplastic mouse mammary tissues from virgin mice.

Experimentally induced breast cancer is often preceded by the appearance of preneoplastic lesions which possess the attributes of hyperplastic normal tissue. These lesions can be isolated and carried as stably transplantable outgrowth lines which continue to morphologically resemble differentiating mammary tissue (Medina, D. Methods Cancer Res., 7: 3-53, 1973). We established seven serially transplantable hyperplastic alveolar nodule (HAN) outgrowth lines from virgin mouse mammary tissues following induction by mouse mammary tumor virus, dimethylbenz(alpha)-anthracene, and/or pituitary isografts. The expression of mammary differentiation-specific casein genes was measured in these hyperplastic outgrowths by immunocytochemistry, specific radioimmune precipitation, and blot hybridization of total RNA. All seven HAN outgrowth lines were immunologically positive for casein both in situ and upon explant culture. Unlike explants from normal virgin mouse mammary gland, exposure to insulin, hydrocortisone, and prolactin induced an increase in casein synthesis in HAN explant cultures which was independent of DNA synthesis. [35S]Methionine-labeled polypeptides synthesized in explant cultures of HAN outgrowths freshly isolated from virgin hosts were analyzed by radioimmune precipitation and gel electrophoresis. This analysis demonstrated that all major species of casein, alpha (Mr 46,000), beta (Mr 27,000), and gamma (Mr 25,000), were constitutively (i.e., in the absence of lactogenic stimuli) expressed in these preneoplastic alveolar mammary outgrowths. In support of this observation, RNA homologous to beta- and alpha-casein cDNA probes was often detectable in total RNA preparations from freshly isolated fragments of HAN outgrowths. A second mammary differentiation specific gene product, alpha-lactalbumin, was also detected in HAN outgrowths both in situ and following explant culture. Enzymatically active alpha-lactalbumin was present in extracts of freshly isolated HAN outgrowth tissues and was detectable in these same tissues by immunoperoxidase. In general, alpha-lactalbumin synthesis was increased during explant culture in the presence of lactogenic hormones; however, in contrast to casein synthesis, insulin-hydrocortisone-prolactin-induced increase in alpha-lactalbumin production in vitro was occasionally dependent upon DNA synthesis as it is in explants from normal virgin mouse mammary tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2004

Rapid preparation of total nucleic acids from E. coli for multi-purpose applications.

Separate protocols are commonly used to prepare plasmid DNA, chromosomal DNA, or total RNA from E. coli cells. Various methods for the rapid preparation of plasmid DNA have been developed previously, but the preparation of the chromosomal DNA and total RNA are usually laborious. We report here a simple, fast, reliable, and cost-effective method to extract total nucleic acids from E. coli by direct lysis of the cells with phenol. Five distinct and sharp bands, which correspond to chromosomal DNA, plasmid DNA, 23S rRNA, 16S rRNA, and a mixture of small RNA, were observed when analyzing the prepared total nucleic acids on a regular 1-2% agarose gel. The simple and high-quality preparation of the total nucleic acids in a single tube allowed us to rapidly screen the recombinant plasmid, as well as to simultaneously monitor the change of the plasmid copy number and rRNA levels during the growth of E. coli in the liquid medium.

Biological Assay↗
PubMedDate not supplied

[Autoradiographic study of RNA synthesis and secretion into the cytoplasm during the early stages of liver regeneration].

Autoradiographic experiments with regeneration of rat liver showed that RNA transport from nucleus to cytoplasm occurs more quickly in 3h following partial hepatectomy while the total RNA synthesis increases insignificantly. Nucleolar RNA synthesis at this time grows in some hepatocytes and decreases in others though it does not differ on the average from the control one. By the 6th h after partial hepatectomy total RNA synthesis significantly increases simultaneously with the acceleration of RNA transport to cytoplasm. Nucleolar RNA synthesis intensifies in all hepatocytes.

Animals↗
PubMed2006

Concentrations of circulating RNA from healthy donors and cancer patients estimated by different methods.

Circulating RNA (cirRNA) was isolated from plasma and cell surface-bound fractions of blood of healthy women and breast cancer patients. RNA samples were DNase treated and quantified by a SYBR Green II assay. Concentrations of RNA sequences of GAPDH, Ki-67 mRNA, and 18S rRNA were measured by real-time quantitative PCR (RT-qPCR) after reverse transcription with random hexamer primers. The obtained data spread over three orders of magnitude for GAPDH and Ki-67 mRNA signals and two orders of magnitude for the copy number of 18S rRNA in blood fractions in both groups. In blood of healthy donors, no correlation was found between the copy number of GAPDH, Ki-67 mRNA, and 18S rRNA and RNA concentrations measured by the SYBR Green II assay. Within the group of breast cancer patients, the concentration GAPDH and Ki-67 mRNA correlated with the concentration of total RNA only in the cell surface-bound fraction; whereas the concentration of 18S rRNA correlated with total RNA in both, the cell surface-bound fraction and blood. The copy number of Ki-67 mRNA correlated with copy numbers of GAPDH mRNA in all fractions of cirRNA of healthy donors and breast cancer patients. A correlation between copy numbers of Ki-67 mRNA and 18S rRNA was found only in cell surface-bound fraction of breast cancer patients. The data described here demonstrate the necessity of searching for more suitable RNA markers in order to estimate total cirRNA concentrations by RT-qPCR, although mRNA of GAPDH could be used for normalization of the level of cancer-specific mRNA among patients.

Biomarkers, Tumor↗
PubMed2004

RNA synthetic activity of glutamate dehydrogenase: determination of enzyme purity, RNA characteristics, and deamination/amination ratio.

The activity of glutamate dehydrogenase (GDH), an important enzyme in carbon and nitrogen metabolism, is routinely assayed by photometry. The RNA synthetic activity of the enzyme provides new technologies for assaying its activity. The enzyme was made to synthesize RNAs in the absence of DNA and total RNA but with different mixes of the four nucleoside triphosphates (NTPs) in order to investigate the RNA characteristics. RNase VI (hydrolyzes base-paired residues) digested the poly (U,A) RNA completely because the U and A residues were evenly distributed to produce many base-paired regions. Therefore, the synthesis of RNA by GDH was by random addition of NTPs. The RNA synthetic activity of the enzyme was at least 50-fold more active in the deamination than in the amination direction, thus providing a robust technology for assay of the enzyme's activity. cDNAs prepared from the RNAs were subjected to restriction fragment differential display polymerase chain reaction analyses. Sequencing of the cDNA fragments showed that some of the RNA synthesized by GDH shared sequence homology with total RNA. Database searches showed that the RNA fragments shared sequence homologies with the G proteins, adenosine triphosphatase, calmodulin, phosphoenol pyruvate (PEP) carboxylase, and PEP carboxykinase, thus explaining the molecular mode of their functions in signal transduction.

Amination↗
PubMed1998

Competitive reverse transcription polymerase chain reaction for quantifying pre-mRNA and mRNA of major acute phase proteins.

Competitive reverse transcription polymerase chain reaction (RT-PCR) is an increasingly used method for quantifying RNA. The technique involves co-amplification from test RNA with an internal standard using common primers in a single reaction. The standard competes for primers and enzyme and it is therefore referred to as a competitor. A RT-PCR polycompetitor for use in quantifying acute phase serum amyloid A, constitutive serum amyloid A, serum amyloid P component, C-reactive protein, apolipoprotein A1, apolipoprotein A2, glyceraldehyde-3-phosphate dehydrogenase and beta-actin mRNAs has been generated and used in quantitative PCR. The polycompetitor was synthesised from ten oligonucleotides of 77-90 bases using primer extension and contains sequences which permit amplification using priming sites that are present in both hnRNA (pre-mRNA) and mRNA. The polycompetitor was cloned into the expression vector pSP64(polyA) and a polycompetitor transcript with a poly(A)-tail sequence at the 3'-end was generated by in vitro transcription. The poly(A)-tail sequence allows the option of performing reverse transcription using oligo(dT) in addition to directed reverse transcription using the specific 3'-reverse PCR primers. cDNA products generated from amplification of the internal polycompetitor standard and endogenous RNA species were separated by capillary electrophoresis and quantified by UV absorbance at 254 nm. Reproducibility was determined to be very high when starting ratios of internal standard and target mRNA are at an approximate equivalence point. Relative standard deviations were less than 5% between independent RT-PCR reactions with the same sample mix of internal standard and total RNA. Applying the method to total RNA samples harvested at various timepoints following cytokine induction of acute phase mRNAs in endothelial cells demonstrated that quantitative readout from the RT-PCR method correlates well with that obtained from Northern-blotting and is at least 100-fold more sensitive. This method will be useful for studying regulation of acute phase proteins in in vitro tissue culture experiments and may also be applied to clinical tissue samples from patients with inflammatory diseases.

Acute-Phase Proteins↗
PubMed2005

Gene expression levels in small specimens from patients detected using oligonucleotide arrays.

Large-scale gene expression profiling using microarray technology is often limited by the amount of tissue or cells available. A number of RNA amplification protocols have been published to overcome this problem. However, additional amplification steps can result in both a 3' bias and poor reproducibility for low abundance transcripts. We performed microarray experiments using HG-U133A GeneChip arrays to ascertain whether less than the recommended amount of RNA can be used, thus avoiding additional amplification steps. In a titration experiment, 2-10 microg of total RNA from a single cryopreserved patient specimen was used to prepare biotinylated cRNA, and the recommended standard amount of 15 microg of each preparation was used for hybridization. Statistical analysis using box plots, correlation coefficients, MvA plots, and concordance percentages revealed almost identical levels of gene expression, independent of the amount of RNA used for target preparation. Most importantly, there was no statistically significant difference when the concordance percentages for low abundance genes were compared, demonstrating that as little as 2 microg of total RNA is sufficient to perform GeneChip analysis.

Child↗