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PubMed2005

Limitations of mRNA amplification from small-size cell samples.

BACKGROUND: Global mRNA amplification has become a widely used approach to obtain gene expression profiles from limited material. An important concern is the reliable reflection of the starting material in the results obtained. This is especially important with extremely low quantities of input RNA where stochastic effects due to template dilution may be present. This aspect remains under-documented in the literature, as quantitative measures of data reliability are most often lacking. To address this issue, we examined the sensitivity levels of each transcript in 3 different cell sample sizes. ANOVA analysis was used to estimate the overall effects of reduced input RNA in our experimental design. In order to estimate the validity of decreasing sample sizes, we examined the sensitivity levels of each transcript by applying a novel model-based method, TransCount. RESULTS: From expression data, TransCount provided estimates of absolute transcript concentrations in each examined sample. The results from TransCount were used to calculate the Pearson correlation coefficient between transcript concentrations for different sample sizes. The correlations were clearly transcript copy number dependent. A critical level was observed where stochastic fluctuations became significant. The analysis allowed us to pinpoint the gene specific number of transcript templates that defined the limit of reliability with respect to number of cells from that particular source. In the sample amplifying from 1000 cells, transcripts expressed with at least 121 transcripts/cell were statistically reliable and for 250 cells, the limit was 1806 transcripts/cell. Above these thresholds, correlation between our data sets was at acceptable values for reliable interpretation. CONCLUSION: These results imply that the reliability of any amplification experiment must be validated empirically to justify that any gene exists in sufficient quantity in the input material. This finding has important implications for any experiment where only extremely small samples such as single cell analyses or laser captured microdissected cells are available.

Analysis of Variance↗
PubMed2002

cDNA array hybridization after laser-assisted microdissection from nonneoplastic tissue.

Differential gene expression can be investigated effectively by cDNA arrays. Because tissue homogenates result inevitably in an average expression of a bulk of different cells, we aimed to combine mRNA profiling with cell-type-specific microdissection. Using a polymerase chain reaction (PCR)-based preamplification technique, the expression profile was shown to be preserved. We modified the existing protocol enabling to apply the total amount of extracted RNA from microdissected cells. A mean amplification factor of nearly 1000 allowed to reduce the demand of initial RNA to approximately 10 ng. This technique was used to investigate intrapulmonary arteries from mouse lungs ( approximately 500 cell equivalents). Using filters with 1176 spots, three independent experiments showed a high consistency of expression for the preamplified cDNAs. These profiles differed primarily from those of total lung homogenates. Additionally, in experimental hypoxia-induced pulmonary hypertension, amplified cDNA from intrapulmonary vessels of these lungs was compared to cDNA from vessels dissected from normoxic lungs. Validation by an alternative method was obtained by linking microdissection with real-time polymerase chain reaction (PCR). As suggested by the array data, nine selected genes with different factors of up-regulation were fully confirmed by the PCR technique. Thus, a rapid protocol is presented combining microdissection and array profiling that demands low quantities of initial RNA to assess reliably cell-type-specific gene regulation even within nonneoplastic complex tissues.

Animals↗
PubMed1998

Transformation-dependent susceptibility of rat hepatic stellate cells to apoptosis induced by soluble Fas ligand.

Cytokine-driven activation of hepatic stellate cells (HSC) in tissue injury and inflammation is a key pathogenetic event in liver fibrogenesis leading to an expanded pool of matrix producing myofibroblasts (MFB) which represent the transformed counterpart of HSC. We hypothesize that expansion of the pool of MFB might also be accomplished by modulation of apoptosis, which plays an opposite and complementary role to mitosis in the cellular homeostasis. We characterized the susceptibility of HSC in primary culture and of MFB in secondary culture to apoptosis induced by the soluble Fas ligand (sFasL) and related the effects to the expression levels of Fas (APO-1/CD95) and some major proapoptotic and contra-apoptotic protooncogenes. MFB showed a dose-dependent apoptotic reaction upon exposure to sFasL as evidenced by a strong increase of nucleosomal DNA fragments, loss of cellular DNA, positive TUNEL reaction, and annexin staining. The effect was found only if protein synthesis (cycloheximide) or RNA synthesis (actinomycin D) were arrested. HSC maintained for various times in primary culture were completely resistant to sFasL in combination with cycloheximide, but in late primary cultures (day 7 onward) an increasing susceptibility to sFasL-mediated apoptosis was developed. By semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and alkaline phosphatase-anti-alkaline phosphatase staining Fas receptor was identified both in HSC and MFB at comparable expression levels. The expression of the contra-apoptotic protooncogenes bcl-2 and bcl-xl was found to be much stronger in early HSC than in late HSC and MFB as shown by ribonuclease protection assay. The expression of bcl-2 was additionally confirmed by semiquantitative RT-PCR and immunoblotting. Proapoptotic bax was found in comparable quantities at the RNA level in HSC and MFB but at the protein level MFB showed increased bax expression. It is concluded that transformation of HSC to MFB is paralleled by an increasing sensitivity to sFasL-mediated apoptosis, which might be related to a strong decrease of bcl-2 and bcl-xl expression, leading to a preponderance of proapoptotic gene expression in MFB. Modulation of apoptotic susceptibility of transforming HSC could be an important complementary pathway in the pathogenesis of fibrosis.

Animals↗
PubMed1998

Molecular virology of hepatitis C virus: an update with respect to potential antiviral targets.

Hepatitis C virus (HCV), a positive-strand enveloped RNA virus, is a major cause of chronic liver disease worldwide. Cis-acting RNA elements and virus-encoded polypeptides required for HCV replication represent attractive targets for the development of antiviral therapies. Internal ribosome entry site-directed translation of HCV genome RNA produces a long polyprotein which is co- and post-translationally processed to yield at least 10 viral proteins. A host signal peptidase is responsible for maturation of the structural proteins located in the N-terminal one-third of the polyprotein. Thus far, four enzymatic activities encoded by the non-structural (NS) proteins have been reported. The NS2-3 region encodes an autoproteinase responsible for cleavage at the 2/3 site. The N-terminal one-third of NS3 functions as the catalytic subunit of a serine proteinase which cleaves at the 3/4A, 4A/4B, 4B/5A and 5A/5B sites, and NS4A is an essential cofactor for some of these cleavages. NS3 also encodes an RNA-stimulated NTPase/RNA helicase at its C terminus, and NS5B has been shown to possess an RNA-dependent RNA polymerase activity. To date, no functions have been reported for NS4B or NS5A in RNA replication, however, NS5A has been implicated in modulating the sensitivity of HCV to interferon. Sequence and structural conservation within the 3' terminal 98 bases of genomic RNA suggest a functional importance in the virus life-cycle and hence another target for antiviral intervention. Recently, HCV infection was shown to be initiated in chimpanzees following intrahepatic inoculation of RNA transcribed from cloned HCV cDNA. The ability to generate large quantities of infectious HCV RNA may facilitate the development of reliable cell culture replication systems useful for the evaluation of antiviral drugs.

Animals↗
PubMed1981

Protein and RNA composition of a mild (W) and a severe (P6) strain of cucumber mosaic virus.

The protein and RNA composition of mild (W) and severe (P6) chlorosis-inducing strain of cucumber mosaic virus (CMV) have been compared. The coat polypeptide of both strains migrated with an apparent molecular weight of 26.9 +/- 0.4 (95%) x 10(3) on SDS polyacrylamide gels. They also co-migrated, and showed no anomaly in SDS binding, on SDS agarose gels. Their amino acid compositions were similar except for small differences in the number of alanine, glycine and proline residues. Analysis by double immuno-diffusion revealed a fast migrating antigen, which showed a reaction of identity between strains, and also a slow migrating antigen which showed a reaction of partial identity. Both CMV-P6 and CMV-W possessed a normal genome complement of RNAs 1 to 4. In addition, an isolate of both strains was found that contained large quantities of satellite RNA 5. However, its presence did not modify symptom severity in tobacco or tomato.

Amino Acids↗
PubMed1990

In vitro and in vivo analysis of the processing and fate of the peptide products of the short proopiomelanocortin mRNA.

Many peripheral tissues express the proopiomelanocortin (POMC) gene as an 800-base mRNA that lacks the 5' end of the 1200-base pituitary transcript. The missing region encodes the peptide signal sequence, and thus, it is unlikely that any translation product would be secreted. We have found that a RNA transcript equivalent to this short message, generated by transcription in vitro from a T7 polymerase promoter, is translatable in a rabbit reticulocyte lysate, generating peptides of 27.5, 22.5, and 15.5 kD. None of these peptides appears to be processed or protected from proteinase-K digestion by a microsomal membrane fraction. In vivo studies were undertaken by transfecting into GH3 cells one of two expression vectors containing sequences that would produce either a full-length mRNA or a short (800-base) mRNA. The neomycin resistance gene was cotransfected with these plasmids, and 30 permanent cell lines were produced after selection in G418. Cell lines containing the full-length RNA secreted large quantities of ACTH and beta-endorphin immunoreactivity, whereas those expressing the short transcript secreted neither of these peptides. However extractable peptide was present in this latter type of cell line, thereby suggesting that the 800-base mRNA was translated, and that no peptide reached the secretory vesicle. These findings raise important questions about the role of peripheral POMC gene expression.

Adrenocorticotropic Hormone↗
PubMed1989

Rapid amplification of complementary DNA from small amounts of unfractionated RNA.

We have combined, in a rapid and straightforward manner, the synthesis and subsequent amplification of individual cDNA sequences from microgram quantities of unfractionated total RNA. Taq1 polymerase, a thermostable DNA polymerase, and Moloney murine leukemia virus (MMLV) reverse transcriptase share similar buffer conditions; these reactions can be performed sequentially, in a single tube, without the need for purification or changes of buffer after the synthesis of cDNA. In this way, nonspecific losses of material are minimized and the required number of cells is reduced. Cell numbers, particularly from human tissues, can be limiting; the requirement for only small amounts of unfractionated RNA makes possible the isolation and characterization of cDNAs from biological materials available in limited quantities. As a demonstration system, we report the rapid synthesis and amplification of cDNA sequences corresponding to the first exon of human immunoglobulin E (IgE). MMLV reverse transcriptase is used with specific (i.e., IgE) or generic (i.e., oligo-[dT(12-18)]) oligomers to prime first strand cDNA synthesis from unfractionated RNA isolated from a human myeloma line, U-266. The necessary primers, deoxynucleotides and Taq1 polymerase, required for second strand cDNA synthesis and the subsequent logarithmic amplification process, are then added to the reaction mixture. This technique provides a useful means of characterizing expressed and processed gene transcripts.

DNA↗
PubMedDate not supplied

[Changes in the chemical composition and physico-chemical characteristics of chromatin from spleens of mice during immunogenesis].

Comparative studies have been carried out for the chemical composition and physico-chemical characteristics of chromatin isolated from spleens of non-immunized and immunized mice. It is found that the chromatin from spleens of immunized mice contains significantly more non-histone proteins and RNA, while the quantity of histone proteins is unaltered. The melting temperatures of chromatin from spleens of non-immunized and immunized mice in 2.5-10(-4) M EDTA (pH 8.0) are 76.8+/-1.50 degrees and 74.4+/-1.10 degrees, respectively. DNA isolated from chromatin melts at 40 degrees. The melting of chromatin was followed in 5 mM sodium-cacodylate buffer (pH 7.0)+1.5-10(-4) M EDTA containing increasing concentrations of urea. The results show that during immunogenesis the changes of the chemical composition of the chromatin are accompanied by certain destabilisation of DNP complex.

Animals↗
PubMed2004

A comparative analysis of data generated using two different target preparation methods for hybridization to high-density oligonucleotide microarrays.

BACKGROUND: To generate specific transcript profiles, one must isolate homogenous cell populations using techniques that often yield small amounts of RNA, requiring researchers to employ RNA amplification methods. The data generated by using these methods must be extensively evaluated to determine any technique dependent distortion of the expression profiles. RESULTS: High-density oligonucleotide microarrays were used to perform experiments for comparing data generated by using two protocols, an in vitro transcription (IVT) protocol that requires 5 microg of total RNA and a double in vitro transcription (dIVT) protocol that requires 200 ng of total RNA for target preparation from RNA samples extracted from a normal and a cancer cell line. In both cell lines, about 10% more genes were detected with IVT than with dIVT. Genes were filtered to exclude those that were undetected on all arrays. Hierarchical clustering using the 9,482 genes that passed the filter showed that the variation attributable to biological differences between samples was greater than that introduced by differences in the protocols. We analyzed the behavior of these genes separately for each protocol by using a statistical model to estimate the posterior probability of various levels of fold change. At each level, more differentially expressed genes were detected with IVT than with dIVT. When we checked for genes that had a posterior probability greater than 99% of fold change greater than 2, in data generated by IVT but not dIVT, more than 60% of these genes had posterior probabilities greater than 90% in data generated by dIVT. Both protocols identified the same functional gene categories to be differentially expressed. Differential expression of selected genes was confirmed using quantitative real-time PCR. CONCLUSION: Using nanogram quantities on total RNA, the usage of dIVT protocol identified differentially expressed genes and functional categories consistent with those detected by the IVT protocol. There was a loss in sensitivity of about 10% when detecting differentially expressed genes using the dIVT protocol. However, the lower amount of RNA required for this protocol, as compared to the IVT protocol, renders this methodology a highly desirable one for biological systems where sample amounts are limiting.

Algorithms↗
PubMed1991

[Quantification of mRNA coding for enterocyte fatty acid binding proteins (FABP) in rats: effect of high lipid diet and starvation].

We have tested in adult rat the specificity and the sensitivity of two cDNA probes derived from recombinant plasmids pJG19 and pJG418. The Northern blot analysis of total RNA extracted from small bowel reveals 0.9 and 0.7 kb mRNA corresponding respectively to the I and L-FABPc mRNA. A linear relation occurs between the amounts of input total RNA and the quantities of specific mRNA found by hybridization with cDNA probes (r = 0.991 and r = 0.998 for I and L-FABPc respectively). We have also evaluated the effect of drastic nutritional status: a high fat diet containing 45% of lipids and a starvation of three days. A two fold increase in I and L-FABPc mRNA occurs in starved rats. By contrast, only the I-FABPc mRNA are slightly enhanced after the hyperlipidic diet (50%). These preliminary results, which must be confirmed by further experiments, suggest a differential nutritional regulation of I and L-FABPc gene expression in rat intestine.

Animals↗
PubMed2003

Total RNA yield and microarray gene expression profiles from fine-needle aspiration biopsy and core-needle biopsy samples of breast carcinoma.

BACKGROUND: Gene expression profiling should be applicable to needle biopsy samples if microarray technology is to become practically useful for clinical research or management of breast carcinoma. This study compared gene expression profiles derived from fine-needle aspiration biopsy (FNAB) and from core needle biopsy (CBX). METHODS: Total RNA was extracted from single FNAB and CBX samples. Corresponding pairs of FNAB and CBX were analyzed for similarity of gene expression profiles using cDNA microarrays that contain 30721 human sequences. A subset of genes that distinguished CBX samples from FNAB samples was evaluated in a larger group of needle biopsy samples and in a published genomic database derived from 78 sporadic breast carcinomas with known clinical outcome. RESULTS: Sixty-eight patients with newly diagnosed breast carcinoma were included in the current study. Sixty-five patients underwent FNAB (17 had both FNAB and CBX) and 3 underwent CBX only. Extracted RNA was of suitable quality for hybridization in 46 (71%) FNABs and 15 (75%) CBXs. Total RNA yield in those samples was similar for single-pass FNAB (mean = 3.6 microg and median = 2.2 microg; n = 46) and CBX (mean = 2.8 microg and median = 2.0 microg; n = 15), with 1 microg or more of total RNA in all cases. Transcriptional profiling was performed successfully in all cases when it was attempted, in a total of 50 samples (38 FNABs and 12 CBXs), including matched FNAB and CBX samples from 10 patients. There were differences in gene expression profiles in 10 matched FNAB and CBX sample pairs. Genes that were expressed differently in CBX samples, compared with FNAB samples, were recognized as being predominantly from the endothelium, fibroblasts, myofibroblasts or smooth muscle, and histiocytes. Corresponding microscopic cell counts from FNABs demonstrated means of 80% tumor cells, 15% lymphocytes, and 5% stromal cells, whereas CBXs contained 50% tumor cells, 20% lymphocytes, and 30% stromal cells. Considering that CBXs are approximately six-fold richer in nonlymphoid stromal cells than FNABs and that CBXs differentially express a set of recognized stromal genes, the authors used these biopsies to define a transcriptional profile of breast carcinoma stroma. A set of 120 genes differentially expressed in CBXs was assessed independently in a published breast carcinoma genomic database to classify breast carcinomas based on stromal gene expression. Subgroups of tumors with low or high stromal signal were identified, but there was no correlation with the development of systemic metastases within 5 years. CONCLUSIONS: Both FNAB and CBX yield a similar quality and quantity of total RNA and are suitable for cDNA microarray analyses in approximately 70-75% of single-pass samples. Transcriptional profiles from FNAB and CBX of the same tumor generally are similar and are driven by the tumor cell population. The authors concluded that each technique has relative advantages. The FNABs provide transcriptional profiles that are a purer representation of the tumor cell population, whereas transcriptional profiles from CBXs include more representation from nonlymphoid stromal elements. Selection of the preferred needle biopsy sampling technique for genomic studies of breast carcinomas should depend on whether variable stromal gene expression is desirable in the samples.

Adult↗
PubMed2006

Molecular monitoring of chronic myelogenous leukemia: identification of the most suitable internal control gene for real-time quantification of BCR-ABL transcripts.

Monitoring breakpoint cluster region-Abelson kinase (BCR-ABL) levels in patients treated for chronic myelogenous leukemia (CML) has become an integral part of patient management. Real-time reverse transcriptase-polymerase chain reaction is the method of choice for this purpose because of its high analytical sensitivity and reproducibility. Given the variation of RNA quality and quantity in clinical specimens, accurate quantitative assessment of BCR-ABL depends on normalization of the BCR-ABL signal to an appropriate internal reference. However, the controls used by different laboratories vary, and there is no clear consensus on an ideal reference due to limited investigations. In this study, we compared nine commonly used control genes for three criteria: mRNA abundance, levels in CML and non-CML cells, and their degradation kinetics in comparison with BCR-ABL. We found that beta-glucuronidase (GUSB) is the most suitable among the nine genes tested. Although ABL is most widely used, our data suggest that the amount of ABL is different in CML and non-CML cells. Moreover, ABL levels are regulated by cellular stress. These findings have a direct impact on current clinical laboratory practice and patient care because the use of a proper control gene affects the reported levels of BCR-ABL transcripts used for patient management decisions.

Adult↗
PubMed1992

Synthesis of backbone deuterium labelled [r(CGCGAAUUCGCG)]2 and HPLC purification of synthetic RNA.

The chemical synthesis of backbone deuterium labelled [r(CGCGAAU*U*CGCG)]2 (U* = [5'-2H]U) is described. An efficient purification procedure was developed using a polymeric reverse phase (PRP) HPLC column at 60 degrees C. This procedure provided pure RNA dodecamer in the multi-milligram quantities (39% overall yield) necessary for dynamics studies using solid-state deuterium NMR. The purification method has been effectively applied to other RNA sequences and will assist biophysical studies which require relatively large quantities of RNA oligomers.

Amides↗
PubMed2004

Optimization of differential display polymerase chain reaction as a bioindicator for the cladoceran Daphnia magna.

Research on toxicant-responsive genes is providing new and important bioindicators for environmental biologists. Identifying genes whose expression is modulated by toxicant exposure provides important clues into the mechanisms underlying toxicity. In addition, toxicant-responsive genes can be developed as molecular end points that are likely to be sensitive tools for environmental assessment. Differential display polymerase chain reaction (ddPCR) is a useful approach for screening and analyzing the expression of genes. A ddPCR protocol was optimized to investigate gene expression in the cladoceran Daphnia magna. The modified protocol requires submicrogram quantities of total RNA (from <10 animals) and utilizes a sensitive fluorescent tagging system. By reverse-transcribing total RNA with arbitrary 18-nucleotide primers and PCR-amplifying the cDNA using the same arbitrary primers under low-stringency conditions, reproducible and consistent ddPCR profiles were generated. Minimal variability was introduced by reaction differences or biological variability. A trial stress (starvation) was found to generate modest differences in the ddPCR profiles. This technique promises to significantly advance knowledge regarding gene expression during toxicant insult. Furthermore, this represents the first step in the development of a novel gene fingerprinting technique that can be applied to any compound and organism of interest.

Animals↗
PubMed1995

A retrospective study of hepatitis C virus carriers in a local endemic town in Japan. A possible presence of asymptomatic carrier.

Chronic hepatitis, cirrhosis, and hepatocellular carcinoma are the accepted sequelae of chronic hepatitis C virus (HCV) infection. However, the real natural history of HCV infection is not still well understood. To approach this problem, we investigated 91 individuals positive for antibodies against HCV (anti-HCV), who have received annual liver function examination in a local town known to have had high carrier rates of hepatitis B virus (HBV) and HCV. Among the 91 anti-HCV-positive individuals, 63 had undertaken the annual examination more than five times in the past 14 years. We analyzed retrospectively the past liver function test results of these 63 subjects and evaluated their present virological status by determining HCV genotypes and estimating quantity of HCV RNA in the sera. Among the 63 subjects, 50 (79.4%) had HCV RNA in the serum and 40 (80%) of the 50 subjects with HCV RNA had abnormal alanine aminotransferase or aspartate aminotransferase level more than once in their records. However, the other 10 (20%) had no abnormal levels during the period examined. Six of 50 (12%) had ultrasonographic findings suggestive of cirrhosis. Thus, HCV-infected individuals in this area did not seem to have progressive liver diseases. Considering the advanced ages of the individuals examined (mean 64 years old), we may have observed a stage in the natural history of HCV infection in which viremia persists in most individuals and the tendency to progress to serious chronic liver disease is mild.

Base Sequence↗
PubMed2006

Extent of over-expression of hepatocyte growth factor receptor in colorectal tumours is dependent on the choice of normaliser.

For reliable results from quantitative RT-PCR, the starting quantity of total RNA and other parameters need to be controlled. Most studies do this by normalising their results to a single reference gene. This study quantified the mRNA expression of three putative reference genes (ubiquitin C, cyclophilin E, and porphobilinogen deaminase) and the target gene hepatocyte growth factor receptor (HGFR) in matched colorectal tumour and normal mucosa samples. Each of the putative reference genes was found to be significantly over-expressed in the tumour samples compared to the normal samples. When HGFR expression was normalised to each of these reference genes using the 2 (-DeltaDeltaC(T)) method of relative quantification, the number of tumour samples in which HGFR was found to be over-expressed varied from 30% to 63% depending on the reference gene chosen for normalisation. This shows that normalising to a single reference gene without prior validation is inappropriate.

Adult↗
PubMed2004

Characterization of an animal model of spontaneous congenital unilateral obstructive uropathy by cDNA microarray analysis.

INTRODUCTION: The pathophysiology of congenital obstructive uropathy has been studied intensively in several animal models. A reliable parameter for early detection of relevant obstruction is not yet identified. For further investigation of the complex regulatory events of obstructive uropathy, we used a cDNA microarray technology for a parallel differential expression analysis of about 15000 different genes of obstructed, contralateral and healthy kidneys of rats with spontaneous congenital obstructive uropathy. METHODS: Total cellular RNA of obstructed, contralateral and healthy control kidneys of 32-35 days old rats was extracted and pooled. RNA quality and quantity was assessed using a Bioanalyzer 2100. mRNA expression for selected marker genes was assessed by real time PCR. High throughput gene expression profiling was performed with cDNA microarrays with differentially labeled cDNA targets. RESULTS: Beside expected typical alterations of several growth factors such as TGF-beta, EGF, IGF-1, PDGF, we observed overexpression of extracellular matrix proteins and a decreased activity of antioxidant enzymes. Additionally, NFkappaB, a nuclear transcription factor involved in the development of interstitial fibrosis, was slightly up-regulated in the obstructed kidneys. Down-regulation of genes involved in tubular sodium transport indicated impaired concentration ability of the obstructed kidneys. Furthermore, we found up-regulation of pro-apoptotic genes including transcripts for the proapoptotic protein Siva. Interestingly, TNFalpha, another factor involved in the pathophysiology of congenital obstructive uropathy, was not differentially regulated. CONCLUSIONS: The alterations of the genes for growth factors, extracellular matrix proteins, antioxidant enzymes, sodium transport and genes involved in apoptosis support the representative character of this animal model for congenital obstructive uropathy. In the rather advanced stage of congenital renal obstruction a possible explanation of the lacking differential regulation of TNFalpha highlights it as a possible marker of earlier stages of obstruction. Furthermore, the possible involvement of the Siva/CD27 pathway in the apoptotic cascade also offers a new possibility to find a sensitive marker to detect renal obstruction already in an earlier stage.

Analysis of Variance↗
PubMed2006

Detection of respiratory syncytial virus and human metapneumovirus by reverse transcription polymerase chain reaction in adults with and without respiratory illness.

BACKGROUND: Reverse transcription polymerase chain reaction (RT-PCR) is a powerful tool that allows the detection of minute quantities of viral RNA. Because of the sensitivity of these assays it is possible that the finding of viral RNA indicates not only active infection but also transient colonization or residual nucleic acid from a distant infection. Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are two viruses for which RT-PCR is now frequently used for diagnosis in adult disease. OBJECTIVE: We evaluated nasal secretions from adults with and without respiratory illnesses by nested, one-tube RT-PCR for RSV and hMPV to determine if rates of detectable RNA were significantly higher among ill subjects compared to controls suggesting a causal relationship with respiratory illness. METHODS: Adults presenting to a health care provider with complaints of respiratory illness were recruited as "cases" and those visiting for non-respiratory complaints were recruited as "controls". Subjects were enrolled during a 3-month period (January to April) when both viruses were expected to be prevalent in the community. Nasal swab samples were obtained and subjected to one-tube nested RT-PCR for RSV and hMPV. RESULTS: Of 146 ill subjects, 17 (11.6%) tested positive for RSV and 5 (3.4%) were positive for hMPV. Of the 158 control subjects, one was RT-PCR positive for RSV and none tested positive for hMPV. The rates of RT-PCR positive cases compared to controls were significantly different for RSV (p<.0001) and hMPV (p<.02). Subjects remained RSV RT-PCR positive on average until day 7.1+/-2.8 of symptoms with a range of 3-10 days. No subject had a positive swab on days 14, 21 or 28. CONCLUSION: Asymptomatic carriage of RSV or hMPV is uncommon. RT-PCR should be a useful method for the diagnosis of these viral illnesses in adults.

Adult↗