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PubMed2005

Quantitative detection of a marine fish iridovirus isolated from large yellow croaker, Pseudosciaena crocea, using a molecular beacon.

A real-time polymerase chain reaction (PCR) assay utilizing a molecular beacon for the quantitative detection of a marine fish iridovirus isolated from large yellow croaker, Pseudosciaena crocea (LYCIV), was developed, which involved the amplification of a 122bp DNA fragment from a conserved region of LYCIV ATPase gene. The specific probe consisting of two short arm and a central loop sequences complementary to the target amplicon was characterized with respect to its efficiency of quenching (E(ff)), and signal to background ratio by spectrofluorometric analysis of its hybridization with the complementary oligonucleotide target. The positive control plasmid pFHT-ATPase containing the target sequence was quantified to make the standard curve for sample detection after serial 10-fold dilution. Linear coefficient correlations between cycle threshold (C(T)) value and logarithmic positive plasmid concentration were close to one (r(2)=0.998) and the detection limit of the assay was 70 copies of positive plasmid/assay. The specificity of this real-time PCR was also demonstrated by using the genomic DNA templates from the healthy fish, white spot syndrome baculovirus (WSSV), and epizootic heamatopietic necrosis virus (EHNV), respectively. The coefficient of variation (CV) of the assay ranged from 1.16 to 4.42%, depending on the concentration of the positive plasmid. The quantitative detection of different tissues from LYCIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (6.86 x 10(6) and 4.62 x 10(6) viral genome copies/mg tissue, respectively) while no viral DNA was detected in the muscular tissue. These results suggested that the real-time PCR assay reported here could be used for rapid, sensitive, and quantitative detection of LYCIV infection.

Animals↗
PubMed2006

Detection and measurement of benzimidazole resistance alleles in Haemonchus contortus using real-time PCR with locked nucleic acid Taqman probes.

Benzimidazole resistance is a common problem in parasitic nematodes of ruminants and early detection is vital if its spread is to be monitored and controlled. Real time PCR offers a fast and reliable method for rapid detection and measurement of resistance allele frequencies. In Haemonchus contortus a single nucleotide polymorphism at codon 200 of the beta-tubulin gene (TTC to TAC), causing a phenylalanine to tyrosine amino acid substitution, has been shown to be involved in many cases of resistance. Locked nucleic acid (LNA) Taqman probes have been used in this work to detect and measure the frequency of resistance alleles in individual and multiple H. contortus. Detection of resistant genotypes using LNA Taqman probes in individual H. contortus is simpler and more reliable than with previously described assays. Measurement of the frequency of resistant alleles in populations of H. contortus was achieved by using the cycle threshold (C(t)) values and a standard curve derived from populations with known allele frequencies. Results using the LNA probes on individual and multiple worms gave similar results to the allele specific PCR. The sensitivity of the LNA assay on multiple nematodes allowed reliable detection of > or = 10% resistance allele frequency. Using the final fluorescence method, it was possible to differentiate populations with approximately 0, 5 and 10% resistance allele frequencies.

Alleles↗
PubMed2000

Simultaneous quantitation of two orchid viruses by the TaqMan real-time RT-PCR.

Simultaneous quantitation of two orchid viruses, cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV), were carried out using the TaqMan((R)) real-time RT-PCR, a novel detection technique that combines RT-PCR with the power of fluorescent detection. Four TaqMan((R)) probes were synthesized, targeting at the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of both viruses. The reporter dye FAM (6-carboxyfluorescein) was used to label the 5' terminus of probes specific to CymMV, while TET (tetrachloro-6-carboxyfluorescein) was used for the ORSV probes. TAMRA (6-carboxy-tetramethyl-rhodamine), which was attached at the 3' terminus of each probe, was used as the universal quencher. With increasing amounts of standard RNA templates, the respective threshold cycle (C(T)) values were determined and a linear relationship was established between these C(T) values and the logarithm of initial template amounts. The amounts of starting templates in mixed-infected Oncidium flowers and leaves were estimated from the standard curves. As little as 10(4) copies or 5 fg each of CymMV and ORSV could be detected simultaneously with either the RdRp or CP gene as the target. This system offers a sensitive, high throughput and rapid method for plant virus detection.

Capsid↗
PubMed2002

Quantitative assay for measuring the Taura syndrome virus and yellow head virus load in shrimp by real-time RT-PCR using SYBR Green chemistry.

Taura syndrome virus (TSV) and yellow head virus (YHV) are the two RNA viruses infecting penaeid shrimp (Penaeus sp.) that have caused major economic losses to shrimp aquaculture. A rapid and highly sensitive detection and quantification method for TSV and YHV was developed using the GeneAmp 5700 Sequence Detection System and SYBR Green chemistry. The reverse transcriptase polymerase chain reaction (RT-PCR) mixture contained a fluorescent dye, SYBR Green, which exhibits fluorescence enhancement upon binding to double strand cDNA. The enhancement of fluorescence was found to be proportional to the initial concentration of the template cDNA. A linear relationship was observed between input plasmid DNA and cycle threshold (C(T)) values for 10(6) down to a single copy of both viruses. To control for the variation in sample processing and in reverse transcription reaction among samples, shrimp beta-actin and elongation factor-1alpha (EF-1alpha) genes were amplified in parallel with the viral cDNA. The sensitivity and the efficiency of amplification of EF-1alpha was greater than beta-actin when compared to TSV and YHV amplification efficiency suggesting that EF-1alpha is a better internal control for the RT-PCR detection of TSV and YHV. In addition, sample to sample variation in EF-1alpha C(T) value was lower than the variation in beta-actin C(T) value of the corresponding samples. The specificity of TSV, YHV, EF-1alpha and beta-actin amplifications was confirmed by analyzing the dissociation curves of the target amplicon. The C(T) values of TSV and YHV samples were normalized against EF-1alpha C(T) values for determining the absolute copy number from the standard curve of the corresponding virus. The method described here is highly robust and is amenable to high throughput assays making it a useful tool for diagnostic, epidemiological and genetic studies in shrimp aquaculture.

Actins↗
PubMed1999

One-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses.

A one-tube reverse transcription-polymerase chain reaction (RT-PCR) for absolute feline coronavirus (FCoV) quantitation was developed. The assay is based on the 5' nuclease activity of the Thermus flavus (Tfl) polymerase and a fluorogenic probe which generates fluorescence when it is cleaved. The fluorogenic probe, also called TaqMan(TM) probe (Perkin Elmer, Foster City, USA), is an oligonucleotide designed to bind between the two PCR primers to the target cDNA and is labeled with a reporter and a quencher dye. In the intact probe, the quencher dye suppresses the fluorescence of the reporter dye by Forster-type energy transfer. During the polymerase extension steps the Tfl exonuclease activity cleaves the hybridised probe resulting in the generation of fluorescent emission of the reporter dye. The threshold cycle (C(T) value) indicates the increase of reporter fluorescence and is directly related to the initial amount of target cDNA or RNA, respectively. Fluorescence is monitored in real time after each cycle by a Perkin-Elmer ABI Prism 7700 Sequence Detector. After completion of amplification, the C(T) values of the samples are calculated back to a standard curve, generated by amplification of diluted standard molecules. The one-tube RT-PCR described below allows precise quantitation, is highly sensitive, rapid (no separate reverse transcription step and no post-amplification steps), easy to handle, allows for a high sample throughput, shows a very good reproducibility, and can be executed with a low risk of contamination. The design of the primers probe combination enables the detection of all known FCoV strains and is also useful for the detection of canine coronavirus, transmissible gastroenteritis virus and porcine respiratory coronavirus.

Animals↗
PubMed2004

RT-PCR analysis of RNA extracted from Bouin-fixed and paraffin-embedded lymphoid tissues.

In the present study, we have investigated whether RNA can be efficiently isolated from Bouin-fixed or formalin-fixed, paraffin-embedded lymphoid tissue specimens. To this aim, we applied a new and simple method that includes the combination of proteinase K digestion and column purification. By this method, we demonstrated that the amplification of long fragments could be accomplished after a pre-heating step before cDNA synthesis associated with the use of enzymes that work at high temperature. By means of PCR using different primers for two examined genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]- and CD40), we amplified segments of cDNA obtained by reverse transcription of the isolated RNA extracted from Bouin-fixed or formalin-fixed paraffin-embedded tissues. Amplified fragments of the expected sizes were obtained for both genes tested indicating that this method is suitable for the isolation of high-quality RNA. To explore the possibility for giving accurate real time quantitative RT-PCR results, cDNA obtained from matched frozen, Bouin-fixed and formalin-fixed neoplastic samples (two diffuse large cell lymphomas, one plasmacytoma) was tested for the following target genes: CD40, Aquaporin-3, BLIMP1, IRF4, Syndecan-1. Delta threshold cycle (DeltaC(T)) values for Bouin-fixed and formalin-fixed paraffin-embedded tissues and their correlation with those for frozen samples showed an extremely high correlation (r > 0.90) for all of the tested genes. These results show that the method of RNA extraction we propose is suitable for giving accurate real time quantitative RT-PCR results.

Acetic Acid↗
PubMed2001

Real-time quantitative PCR analysis for alpha-thalassemia-1 of Southeast Asian type deletion in Taiwan.

Since homozygosity of the alpha-thalassemia-1 of Southeast Asian (SEA) type deletion results in hydrops fetalis, a novel protocol based on the real-time quantitating polymerase chain reaction (PCR) technique has been developed to quantify the intact and aberrant alpha-globin genes in adults. The ratio of the normal/SEA-bearing alpha-globin genes was expressed in cycle threshold (C(T)) values. Theoretically, a relative ratio of one to one was anticipated in individuals carrying the SEA type deletion. Twenty-five heterozygous and 20 normal cases were analyzed retrospectively with this protocol. Data showed that the CT values for the intact alpha-globin gene allele and the allele bearing the SEA type deletion in carriers were 28.74+/-1.49 and 26.46+/-2.05, respectively. Therefore, the ratio of normal/SEA type deletion-bearing alpha-globin genes in the carriers was 1.09+/-0.043. No ambiguous results were observed from other less common genotypes associated with alpha-thalassemia, such as the Philippine type deletion. Based on the results, we concluded that this protocol could provide a rapid method to mass screen carriers with alpha-thalassemia-1 of SEA type deletion in this region.

Alleles↗
PubMed2003

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR.

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.

Colony Count, Microbial↗
PubMed2004

Real-time PCR quantitation of clostridia in feces of autistic children.

Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children. We applied the TaqMan real-time PCR procedure to detect and quantitate three Clostridium clusters and one Clostridium species, C. bolteae, in stool specimens. Group- and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains. In this procedure, a linear relationship exists between the threshold cycle (CT) fluorescence value and the number of bacterial cells (CFU). The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell of C. bolteae could be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children for C. bolteae and the following Clostridium groups were statistically significant: mean counts of C. bolteae and clusters I and XI in autistic children were 46-fold (P = 0.01), 9.0-fold (P = 0.014), and 3.5-fold (P = 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 x 10(8) CFU/g in autistic children and 4.8 x 10(8) CFU/g in controls; respectively). More subjects need to be studied. The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract.

Autistic Disorder↗
PubMed2005

Real-time PCR detection of Vibrio vulnificus in oysters: comparison of oligonucleotide primers and probes targeting vvhA.

We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (C(T)) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 10(3) V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 x 10(3) CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.

Animals↗
PubMed2006

Evaluation of real-time and conventional PCR targeting complex 85 genes for detection of Mycobacterium leprae DNA in skin biopsy samples from patients diagnosed with leprosy.

In spite of the decrease in the number of registered leprosy patients, the number of new cases diagnosed each year (400,000) has remained essentially unchanged. Leprosy diagnosis is difficult due to the low sensitivity of current methodologies to identify new cases. In this study, conventional and TaqMan real-time PCR assays for detection of Mycobacterium leprae DNA were compared to current classification based on clinical, bacteriological, and histological evaluation. M. leprae DNA was extracted from frozen skin biopsy specimens from 69 leprosy patients enrolled in the study and was amplified using specific primers for either the antigen 85B-coding gene or the 85A-C intergenic region by using conventional and real-time PCR. The detection rate was 100% among multibacillary (MB) patients and ranged from 62.5% to 79.2% among paucibacillary (PB) patients according to the assay used. The TaqMan system for 85B gene amplification showed the highest sensitivity, although conventional PCR using the 85A-C gene as a target was also efficient. The cycle threshold (C(T)) values obtained using the TaqMan system were able to statistically (P < 0.0001) differentiate MB (mean C(T), 28.06; standard deviation [SD], 4.51) from PB (mean C(T), 33.06; SD, 2.24) patients. Also, there was a correlation between C(T) values and the bacteriological index for MB patients (Pearson's r, -0.444; P = 0.008). Within the PB patients' group, we tested normal skin from six patients exhibiting the pure neuritic form of leprosy (PNL). Five out of six PNL patients were positive for the presence of M. leprae DNA, even in the absence of skin lesions. In conclusion, the TaqMan real-time PCR developed here seems to be a useful tool for rapidly detecting and quantifying M. leprae DNA in clinical specimens in which bacilli were undetectable by conventional histological staining.

Antigens, Bacterial↗
PubMed2001

Detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR Green chemistry.

A rapid and highly sensitive real-time PCR detection and quantification method for infectious hypodermal and hematopoietic necrosis virus (IHHNV), a single-stranded DNA virus, and white spot virus (WSV), a double-stranded DNA (dsDNA) virus infecting penaeid shrimp (Penaeus sp.), was developed using the GeneAmp 5700 sequence detection system coupled with SYBR Green chemistry. The PCR mixture contains a fluorescence dye, SYBR Green, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. A linear relationship was observed between the amount of input plasmid DNA and cycle threshold (C(T)) values over a range of 1 to 10(5) copies of the viral genome. To control the variation in sampling and processing among samples, the shrimp beta-actin gene was amplified in parallel with the viral DNA. The C(T) values of IHHNV- and WSV-infected samples were used to determine absolute viral copy numbers from the standard C(T) curves of these viruses. For each virus and its beta-actin control, the specificity of amplification was monitored by using the dissociation curve of the amplified product. Using genomic DNA as a template, SYBR Green PCR was found to be 100- to 2000-fold more sensitive than conventional PCR, depending on the virus, for the samples tested. The results demonstrate that SYBR Green PCR can be used as a rapid and highly sensitive detection and quantification method for shrimp viruses and that it is amenable to high-throughout assay.

Animals↗
PubMedDate not supplied

Effects of short-term cycling on knee joint proprioception in healthy young persons.

BACKGROUND: Criteria are needed for measuring the effects of exercise and fatigue on proprioception. PURPOSE: To measure knee joint proprioception in healthy subjects before and after exercise and to establish a reference for further comparisons of patients with knee injuries. STUDY DESIGN: Controlled laboratory study. METHODS: We tested proprioception in the knees of 24 healthy subjects with a mean age of 24 years and median Tegner score of 5. Subjects were tested to estimate their thresholds for detecting slow passive motion, from starting positions of 20 degrees and 40 degrees before and after cycling on an ergometer bicycle until the pulse rate reached a steady state level and they reached a score of 14 to 17 on Borg's Ratio of Perceived Exertion scale. RESULTS: After cycling, significantly higher threshold values were found for perception of movement toward flexion from both 20 degrees and 40 degrees. No significant differences were seen in measurements of movement toward extension. CONCLUSIONS: Knee joint proprioception seems to be impaired by exercise or training. CLINICAL RELEVANCE: This impairment may lead to defective dynamic stabilization of the joint, leading to an increased risk of injuries.

Adult↗
PubMed2004

Differentiation of acute myeloid leukemia from B- and T-lineage acute lymphoid leukemias by real-time quantitative reverse transcription-PCR of lineage marker mRNAs.

BACKGROUND: Flow cytometry of lineage markers is useful in the classification of leukemias. Our aim was to assess whether the study of lineage genes at the RNA level would enable differentiation of acute myeloid leukemias (AMLs) from B-and T-lineage acute lymphoid leukemias (ALLs). METHODS: We measured mRNA of four lineage markers [CD19, CD79a, CD3e, and myeloperoxidase (MPO)] by reverse transcription followed by real-time quantitative (RTQ)-PCR. We investigated 72 acute leukemias (40 AMLs with 23-93% blast cells plus 27 B-lineage ALLs and 5 T-lineage ALLs) defined by morphologic criteria at diagnosis. RTQ-PCR analysis was performed on bone marrow without cell sorting. The expression of each gene was calculated as the difference in the threshold cycle [DeltaCT; CT value of target gene minus CT value of housekeeping gene (Abelson)]. RESULTS: Three patterns of expression were detected. In the first, CD19, CD79a, and MPO mRNAs were less abundant than CD3e. In the second pattern, MPO mRNA was more abundant than the other three mRNAs. In the third, CD19 or CD79a was more highly expressed than CD3e and MPO. The three patterns corresponded to T-ALL, AML, and B-ALL, respectively. The use of cutoffs to establish qualitatively the pattern of coexpression of the four lineage markers provided the same information as the comparison among the four DeltaCT values. Prospective use of the scoring system correctly classified each of 13 additional cases (8 AML, 4 B-lineage ALL, and 1 T-lineage ALL). CONCLUSION: Study of lineage markers at diagnosis by RTQ-PCR allows differentiation of AML from B-ALL or T-ALL without cell sorting, even when the bone marrow contains few blast cells.

Adult↗
PubMed2002

Threshold disorder as a source of diverse and complex behavior in random nets.

We study the diversity of complex spatio-temporal patterns in the behavior of random synchronous asymmetric neural networks (RSANNs). Special attention is given to the impact of disordered threshold values on limit-cycle diversity and limit-cycle complexity in RSANNs which have 'normal' thresholds by default. Surprisingly, RSANNs exhibit only a small repertoire of rather complex limit-cycle patterns when all parameters are fixed. This repertoire of complex patterns is also rather stable with respect to small parameter changes. These two unexpected results may generalize to the study of other complex systems. In order to reach beyond this seemingly disabling 'stable and small' aspect of the limit-cycle repertoire of RSANNs, we have found that if an RSANN has threshold disorder above a critical level, then there is a rapid increase of the size of the repertoire of patterns. The repertoire size initially follows a power-law function of the magnitude of the threshold disorder. As the disorder increases further, the limit-cycle patterns themselves become simpler until at a second critical level most of the limit cycles become simple fixed points. Nonetheless, for moderate changes in the threshold parameters, RSANNs are found to display specific features of behavior desired for rapidly responding processing systems: accessibility to a large set of complex patterns.

Algorithms↗
PubMed1998

Definition of the potentially fertile period from urinary steroid excretion rates. Part II. A threshold value for pregnanediol glucuronide as a marker for the end of the potentially fertile period in the human menstrual cycle.

Application of time series analysis to a database containing serial pregnanediol data from 113 complete ovulatory menstrual cycles contributed by 83 women of proven fertility and 68 cycles for which pregnanediol values were available over the ovulatory period, detected the first statistically significant risk in pregnanediol excretion for all cycles for which a baseline was available (n = 170). However, even at the 99% confidence level, for 22% of cycles a rise was observed before the presumed day of ovulation. Therefore, a threshold value for pregnanediol was sought from the database as a better marker for the end of fertility. A value of 1.4 mg per 24 h was not reached before day 2 after the pre-ovulatory estrogen peak day for 96% of the cycles. In the remaining 4% of cycles it was reached one day after the total estrogen peak day. The validity of this threshold was confirmed in extensive studies using the Ovarian Monitor where the equivalent is 6.3 mumol per 24 h of pregnanediol glucuronide and measurements are performed on timed urine specimens with a minimum collection time of three hours. These studies were as follows: 1) a World Health Organization study on the use of the Ovarian Monitor as a fertility self test in the home (108 cycles), 2) a multicenter study on returning fertility during breast feeding conducted by Family Health International (73 women), and 3) the general application of the Ovarian Monitor for pregnancy achievement and avoidance during the past ten years (over 250,000 PdG assays performed in ten countries). With rare exceptions, the use of these threshold values is applicable for all women provided correction is made for urine volume.

Biomarkers↗
PubMed2002

Molecular staging of lung cancer: real-time polymerase chain reaction estimation of lymph node micrometastatic tumor cell burden in stage I non-small cell lung cancer--preliminary results of Cancer and Leukemia Group B Trial 9761.

OBJECTIVE: The 5-year survival for patients with surgically resected stage I non-small cell lung cancer is only 60% to 70%, probably because of undetected systemic occult micrometastases. Detection of occult micrometastases in lymph nodes by reverse-transcriptase polymerase chain reaction for carcinoembryonic antigen messenger RNA in non-small cell lung cancer has not been reported. Detection of occult micrometastases by standard reverse-transcriptase polymerase chain reaction provides only yes or no answers about their presence, whereas quantitative real-time reverse-transcriptase polymerase chain reaction permits reproducible quantitation of target molecules. This study evaluated the ability of quantitative reverse-transcriptase polymerase chain reaction to quantitate lymph node occult metastases with carcinoembryonic antigen messenger RNA as a tumor marker. METHODS: Standard reverse-transcriptase polymerase chain reaction and quantitative reverse-transcriptase polymerase chain reaction for carcinoembryonic antigen messenger RNA were performed on 232 lymph nodes from 53 patients with stage I disease (node negative according to histologic examination). Quantitative reverse-transcriptase polymerase chain reaction determined carcinoembryonic antigen messenger RNA quantity by detecting fluorescence increase at a threshold polymerase chain reaction cycle. Threshold polymerase chain reaction cycle values were correlated with standard curves created from serially diluted carcinoembryonic antigen-positive HTB-174 tumor cells to estimate the number of micrometastatic tumor cells in a lymph node. RESULTS: Detection rates of occult metastases were similar for standard reverse-transcriptase polymerase chain reaction and quantitative reverse-transcriptase polymerase chain reaction at 38 of 232 (16.4 %) and 59 of 232 (25.4 %), respectively. Upstaging rates among 53 cases of stage I non-small cell lung cancer were also similar for standard reverse-transcriptase polymerase chain reaction and quantitative reverse-transcriptase polymerase chain reaction at 23 of 53 (43.4 %) and 30 of 53 (56.6%), respectively. Comparison of positive lymph node stations according to quantitative reverse-transcriptase polymerase chain reaction (threshold polymerase chain reaction cycle <45) with HTB-174 tumor cell standard curves yielded estimates of metastatic tumor cell burden of 1.07 x 10(3)to 3.24 x 10(5)cells per lymph node station (median 7190 tumor cells per lymph node station). CONCLUSIONS: Standard and quantitative real-time reverse-transcriptase polymerase chain reaction for carcinoembryonic antigen detected occult metastases in patients with stage I non-small cell lung cancer at similar rates; both upstaged about 50% of cases. Quantitative reverse-transcriptase polymerase chain reaction allows estimation of the number of metastatic cells per lymph node, however, which potentially allows greater precision in predicting recurrence risk.

Adenocarcinoma↗
PubMed2002

A reverse transcription comparative real-time PCR method for quantitative detection of angiogenic growth factors in head and neck cancer patients.

OBJECTIVES: Head and neck cancer is one of the ten most frequent cancers in the world. The angiogenic growth factors VEGF, PDGF and bFGF play a role in cancer aggressiveness. We developed a sensitive method to quantify the gene expression of these factors in the tissues of head and neck cancer patients. DESIGN AND METHODS: All assays were performed using real-time RT-PCR, which yields a value (Ct) denoting the threshold cycle of PCR amplification at which product is first detected by fluorescence. The Ct is dependent on the quantity of the target molecule in the sample. To control for variation in RNA quantity and quality, we used 18S ribosome RNA as an internal control to calculate a relative Ct for the target molecules of interest, VEGF, PDGF and bFGF. A serially diluted positive control sample was analyzed by linear regression to determine the sensitivity and linearity of the assay. Paired normal and cancerous tissue samples from 115 head and neck cancer patients were assayed to ascertain the relative levels of the growth factors. RESULTS: The CVs of within-run and between-run assays for VEGF, PDGF and bFGF were all less than 3%. The correlation coefficient of the RNA concentrations and Ct values were 0.9987, 0.9977, and 0.9996 respectively for VEGF, PDGF and bFGF. The assay was sensitive to as little as 10(-3) ng of RNA. All three growth factors were significantly increased in tumor tissue as compared to normal tissue. VEGF, PDGF and bFGF levels were elevated in 71.3%, 58.2% and 54.0% of cancerous tissue samples, with average levels of over-expression of 35.1, 24.6 and 13. sixfold, respectively. CONCLUSION: This method provides sensitive, quantitative, high-throughput analysis for direct comparison of gene expression levels between samples, while adjusting for factors that may influence quantity determination. It should be applicable to molecules other than angiogenic growth factors, as well.

Endothelial Growth Factors↗