Real-time Polymerase Chain Reaction Technology Research Articles

Comparison of the COBAS/Ampliprep Taqman and Amplicor HIV-1 monitor tests in Lagos, Nigeria.

BackgroundThe use of real-time Polymerase chain reaction (PCR) technology options is increasing in resource-limited settings because they are faster, improve assay sensitivity, have higher throughput, larger dynamic ranges and reduced rates of contamination. In 2010, UNAIDS ranked Nigeria as the second highest population of people living with HIV and AIDS (2.98 million people) in the world.ObjectiveThe objective of this study was to compare the analytical performances of the Amplicor HIV-1 Monitor (version 1.5) and the COBAS Ampliprep/Taqman (version 2.0) used in monitoring HIV disease progression in HIV-infected individuals.MethodIn a cross-sectional study, HIV-1 RNA values obtained with the Amplicor HIV-1 monitor version 1.5 were compared with those of the COBAS/Ampliprep TaqMan HIV-1 version 2.0 in a routine clinical setting. Between May and November 2011, 176 plasma samples collected were analysed in parallel using both techniques. Data analysis was done using statgraphics Centurion XVI and Medcalc version 12.0.ResultThe correlation coefficient for the two assays was 0.83 and the level of agreement using a Bland–Altman plot was 94.2%.ConclusionThese findings suggest that the results from the two methods were comparable, hence the COBAS/Ampliprep Taqman version 2.0 is recommended for high-volume laboratories.

Cecum ligation and dissection: a novel modified mouse sepsis model

BackgroundThe fact that many sepsis therapeutics failed to be translated into the human indicates that there is still a serious need to reassess our models of sepsis research. We aimed to develop a novel modified model of sepsis in the mouse, which simulates the clinical situation more accurately. Materials and methodsSepsis was induced in C57Bl/6 mice by dissecting the cecum and placing the discontinued organ back into the abdomen (cecum ligation and dissection [CLD]). Septic animals were relaparotomized after 6 h, followed by peritoneal lavage, and antibiotic treatment. Results were compared with shams or the classic colon ligation and puncture (CLP) model. The postoperative lung impairment was assessed using neutrophil invasion as a surrogate. Proinflammatory cytokines were measured by either real-time polymerase chain reaction or Luminex technology, and liver damage was evaluated by aspartate transaminase and alanine transaminase measurements. ResultsIn CLD animals with relaparotomy after 6 h, lung interleukin (IL) 6, monocyte chemoattractant protein (MCP)-1 messenger RNA levels, and neutrophil invasion were significantly increased. Liver messenger RNA expression in CLD animals was significantly upregulated for IL-6, tumor necrosis factor alpha, IL-10, and MCP-1 compared with sham and CLP animals. Significantly higher levels of alanine transaminase were observed in CLD animals. Finally, systemic inflammation as measured by plasma IL-6, tumor necrosis factor alpha, IL-1β, IL-10, and MCP-1 was significantly increased in all CLD animals compared with shams, whereas CLP animals only showed an insignificant increase in the latter molecules. ConclusionsOur modifications to the classic CLP model significantly produced organ inflammation, liver damage, and a similar mortality compared with a clinical setting, with a reliable onset of sepsis.

Clinical evaluations of cell‐free fetal DNA quantities in pre‐eclamptic pregnancies

Quantitative changes of cell-free fetal DNA in maternal plasma as an indicator for impending pre-eclampsia was reported in different studies. Cell-free fetal nucleic acids can be detected in maternal circulation during pregnancy. Our aim was to determine the higher rate of fetal DNA levels in maternal blood in pre-eclampsia compared to normal pregnancies and the clinical use of real-time polymerase chain reaction (PCR) in the Turkish population as a marker. According to their gestational ages, the plasma levels of 30 pre-eclamptic women at 26-40 weeks of pregnancy were matched with 18 healthy pregnant women. Cell-free fetal DNA levels in maternal plasma were compared using real-time PCR technology. For the quantitative measurement of fetal DNA from maternal blood, the relative quantification PCR process was applied to all samples, using SRY and GAPDH genes. These patients were classified as pre-eclamptic and control groups and were matched according to weeks of pregnancy. Free fetal DNA levels of 30 pre-eclamptic patients were compared to healthy pregnant women and an average 3.06-fold increase was observed. During the second trimester, free fetal DNA levels were 1.5 times higher in pre-eclamptic patients. This increase was 3.5-fold during the third trimester. The DNA increase of pre-eclamptic patients was 4.1-fold and 3.4-fold during 29th-33rd and 34th-40th weeks, respectively. Cell-free fetal DNA in maternal blood could be used as a marker for identifying subjects at increased risk of developing pre-eclampsia.

Beta-catenin mediates the apoptosis induction effect of celastrol in HT29 cells

AimWe evaluated the apoptosis induction effects of celastrol in human colorectal cancer cell line HT29 in WNT/beta-catenin pathway. Main methodsHT29 cells were treated with various concentrations (10–100μM) for 24h, MTT assay was performed to examine the effect of celastrol on growth inhibition of HT29 cells. Beta-catenin siRNA was used for transfection of cells. Cell apoptosis was detected through both DNA laddering analysis and Tdt-mediated dUTP nick end labeling (TUNEL) assay. Western blot analysis and real-time reverse transcription polymerase chain reaction technologies were applied to assess the expression level of c-Myc, Bax, and Bcl-2 in HT29 cells. Key findingsTreatment of HT29 cells with celastrol resulted in a growth inhibition effect, and the IC50 value was 56μM. Celastrol induced HT29 cells apoptosis, and increased the nuclear translocation of beta-catenin. Apoptosis induction effects of celastrol were significantly attenuated by beta-catenin siRNA transfection. Beta-catenin siRNA markedly increased mRNA and protein levels of c-Myc compared with control siRNA. Beta-catenin siRNA significantly inhibited the expression of Bax and Bcl-2 in celastrol-treated HT29 cells. SignificanceBeta-catenin mediates the apoptosis induction effects of celastrol in HT29 cells.

Impacto clínico de la cuantificación de la carga mutacional de JAK2V617F en pacientes con neoplasias mieloproliferativas crónicas Ph negativas clásicas

Background and objectivesOur study has investigated the presence of the mutation V617F in the JAK2 gene in patients diagnosed with chronic myeloproliferative neoplasms (MPNs). Furthermore, we determined if JAK2 (V617F) allelic burden associates with a specific clinical phenotype and if its quantification can be used as a marker to predict outcome and complications in patients with MPNs. Patients and methodsA retrospective observational study was conducted from 1987 to 2011 in the Haematology Department of La Paz University Hospital. The allelic burden was measured in 114 patients diagnosed with MPNs: 39 polycythemia vera (PV) patients, 71 essential thrombocythaemia patients and 6 primary myelofibrosis patients. The quantitative real-time polymerase chain reaction (qRT-PCR) technology was used to determinate the percentage of mutated alleles in peripheral blood neutrophils. Patients were divided in 2 groups: heterozygous if the result was≤50% of the tested cells, and homozygous if it was positive in>50% of the cells. ResultsSixty-nine patients were positive for the JAK2 mutation and 45 were negative. Among those positive, the mutation was associated with arterial thrombosis. In addition, we demonstrate in the homozygous group that the V617F mutation is associated to PV, advanced age, leukocytosis, marked haematopoiesis and splenomegaly. ConclusionsThe presence of V617F mutation is associated with a higher incidence of thrombosis, leukocytosis and splenomegaly. The identification of mutation on the JAK2 gene could help in a better definition of evolution and prognostic stratification of the myeloproliferative disorders.

Case report

There are several different methods of testing cattle for persistent infection with bovine viral diarrhea virus (PI-BVDV), including immunohistochemistry (IHC), polymerase chain reaction (PCR), virus isolation, and antigen capture enzyme-linked immunosorbent assay (ACE). The purpose of this report is to describe a case in which a pooled PCR test (28 specimens/pool) was compared for sensitivity against an ACE test (tested as single specimens). Ear notch specimens (fresh) were collected from beef calves purchased by a stocker operation. From January through March 2010, this stocker operation received 2,424 calves and took two ear-notch specimens from each calf for PI-BVDV testing. One specimen from each calf was sent to a commercial PI-BVDV testing laboratory that utilizes real-time PCR technology in specimen pools of 28 (Laboratory A), and a second specimen was sent to a commercial PI-BVDV testing laboratory that utilizes ACE (NS23) technology using non-pooled specimens (Laboratory B). Laboratory A detected four positive specimens out of the 2,424 specimens submitted (0.165% PI prevalence), while Laboratory B detected 12 positive specimens out of the 2,424 specimens submitted (0.495% PI prevalence). The 12 specimens detected by Laboratory B included the same four detected by Laboratory A, as well as eight additional positive specimens. Upon receiving these discordant results, the attending veterinarian requested that the 12 positive specimens detected by Laboratory B be re-tested using the original specimens. These original 12 ACE-positive specimens were then re-tested as single specimens at Laboratory B using a commercially available ACE test (Erns) and real-time PCR technology. The 12 original ACE-positive specimens were again positive by these different tests. Follow-up testing on the 12 calves was conducted more than 30 days after initial testing with a second set of samples from the eight surviving calves (four of the 12 calves died between the original and the second collection). The second set of samples from the eight surviving calves initially ACE-positive by Laboratory B were tested using serum, formalin-fixed ear notches, and fresh ear notches in phosphate buffered saline. All specimens were positive by gel-based reverse transcriptase PCR on serum and ear notches (non-pooled), and all eight formalin-fixed notches were also positive by IHC.

NPSR1 Gene Is Associated with Reduced Risk of Rheumatoid Arthritis

Neuropeptide S receptor 1 (NPSR1) is a G protein-coupled receptor involved in immune response and is associated with several inflammatory diseases. We investigated the possible contribution of several polymorphisms in the intronic region of NPSR1 to rheumatoid arthritis (RA). Genotyping of 7 single-nucleotide polymorphisms (SNP) was performed in a total of 1232 patients with RA and 983 healthy controls of Spanish white origin by real-time polymerase chain reaction technology, using the TaqMan 5'-allele discrimination assay. One out of the 7 SNP analyzed (rs740347) was associated with RA [p after Bonferroni correction (p(BNF)) = 1.2 × 10(-3), OR 0.73]. An association was also observed with rheumatoid factor-positive and shared epitope-positive RA (p(BNF) = 0.011, OR 0.73; p(BNF) = 0.037, OR 0.75, respectively). Our results show that variations in the NPSR1 intronic region are associated with low risk in patients with RA, supporting other evidence that this locus represents a common genetic factor in inflammatory diseases.

Factors associated with a strictly undetectable viral load in HIV‐1‐infected patients

The aim of the study was to identify factors associated with a strictly undetectable viral load (VL) using a routine sensitive real-time polymerase chain reaction (RT-PCR) technology. From a large prospective cohort, 1392 patients with a VL<50 HIV-1 RNA copies/mL while receiving a three-drug suppressive regimen for at least 1 year were included in a cross-sectional analysis. Patients were classified into three groups and compared by univariate and multivariate analysis: 479 patients with a strictly undetectable VL (group 1; 34%), 617 patients with detectable VL below the threshold of 20 copies/mL (group 2; 44%), and 296 patients with a VL of 20-50 copies/mL (group 3; 12%). Comparing groups 1 and 2, VL zenith<5 log(10) copies/mL [odds ratio (OR) 1.51; 95% confidence interval (CI) 1.15-1.99; P=0.003], current CD4 T-cell count<500 cells/μL (OR 1.44; 95% CI 1.08-1.92; P=0.01), and duration of viral suppression<50 copies/mL longer than 2 years (OR 2.32; 95% CI 1.20-4.54; P=0.01) were associated with undetectable VL. Comparing groups 1 and 3, VL zenith<5 log(10) copies/mL (OR 2.48; 95% CI 1.75-3.50; P<0.001), duration of viral suppression<50 copies/mL longer than 1 year (OR 3.33; 95% CI 1.66-6.66; P=0.0006), and nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens (OR 1.45; 95% CI 1.03-2.04; P=0.03) were associated with undetectable VL. No individual drug effect was found within NNRTI molecules. Longer duration of viral suppression<50 copies/mL, lower viral load zenith and NNRTI-based regimen were independently associated with a strictly undetectable viral load. This routinely used RT-PCR assay may prove to be a valuable tool in further large-scale studies.

Identification of tuna species by a real-time polymerase chain reaction technique

Tuna are highly priced fishes that are often used in processed products. For effective fishery management and protection of consumers’ rights, it is important to develop a molecular method to identify the species of the tuna products. In this study we have developed a molecular method based on real-time polymerase chain reaction (real-time PCR) technology for the rapid identification of four tuna species. Four species-specific TaqMan probes were designed to identify bigeye tuna (Thunnus obesus), Pacific bluefin tuna (Thunnus orientalis), southern bluefin tuna (Thunnus maccoyii), and yellowfin tuna (Thunnus albacares). A SYBR green system was also designed to enhance the authentication of T. obesus. Both systems can distinguish target species from others in an efficient and high-throughput manner and can be applied to species identification of tuna products.

ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq) data

BackgroundMeasuring gene transcription using real-time reverse transcription polymerase chain reaction (RT-qPCR) technology is a mainstay of molecular biology. Technologies now exist to measure the abundance of many transcripts in parallel. The selection of the optimal reference gene for the normalisation of this data is a recurring problem, and several algorithms have been developed in order to solve it. So far nothing in R exists to unite these methods, together with other functions to read in and normalise the data using the chosen reference gene(s).ResultsWe have developed two R/Bioconductor packages, ReadqPCR and NormqPCR, intended for a user with some experience with high-throughput data analysis using R, who wishes to use R to analyse RT-qPCR data. We illustrate their potential use in a workflow analysing a generic RT-qPCR experiment, and apply this to a real dataset. Packages are available from http://www.bioconductor.org/packages/release/bioc/html/ReadqPCR.htmland http://www.bioconductor.org/packages/release/bioc/html/NormqPCR.htmlConclusionsThese packages increase the repetoire of RT-qPCR analysis tools available to the R user and allow them to (amongst other things) read their data into R, hold it in an ExpressionSet compatible R object, choose appropriate reference genes, normalise the data and look for differential expression between samples.

Comparison of a TaqMan real-time polymerase chain reaction assay with a loop-mediated isothermal amplification assay for detection of Gallid herpesvirus 1

A TaqMan real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assay were developed to detect Gallid herpesvirus 1 (GaHV-1, formerly Infectious laryngotracheitis virus). The standard curve of real-time PCR was established, and the sensitivity reached 10 copies/μl. In the current study, the conversion between viral titer and GaHV-1 genomic copy number was constructed. Six primers for LAMP assay amplified target gene at 65°C within 45 min, and the detection limit was 60 copies/μl. The 6 primers were highly specific, sensitive, and reproducible for detection of GaHV-1. Although the sensitivity of LAMP was lower than that of real-time PCR, LAMP was faster, less expensive, and did not require a thermocycler. The LAMP assay would be a viable alternative assay in diagnostic laboratories that do not employ real-time PCR technology.

Molecular cloning, expression analysis and sequence prediction of CCAAT/enhancer-binding protein beta gene of Qinchuan cattle

CCAAT/enhancer-binding protein beta (C/EBPβ), as an essential transcriptional factor, regulates the differentiation of adipocytes and the deposition of fat. Herein, we cloned the whole open reading frame (ORF) of bovine C/EBPβ gene and analyzed itsputative protein structures via DNA cloning and sequence analysis. Then, theexpression profile of this C/EBPβ gene in fifteen kinds of tissues of Qinchuan cattlewas conducted by real-time polymerase chain reaction (RT-PCR) technology. One significant outside to inside transmembrane structure located in amino acid region from 190 to 208 was observed in the putative protein sequences. Besides, one basic leucine zipper domain (bZIP) in amino acid area from 274 to 337 was found, concurring with the main characteristic of C/EBPs. Homologous comparison of the amino acid sequences from C/EBPβ cloned in this study and those from different species indicated C/EBPβ gene of Qinchuan cattle shared 97, 95 and 91% similarity with Homo sapiens, Sus scrofa and Oryctolagus cuniculus respectively, indicating a good sequence evolutional conservation of C/EBPβ. RT-PCR results revealed bovine C/EBPβ gene mRNA expression level of subcutaneous fat was the highest among all the analyzed tissues, and the relative quantity (RQ) in fat tissue increased as cattle grew. All in all, the present results could be used as basic but important genetic resource and information to inspire additional and specific studies on Qinchuan cattle. Key words: CCAAT/enhancer-binding protein beta, molecular cloning, expression analysis.

Residual DNA analysis in biologics development: Review of measurement and quantitation technologies and future directions

Residual DNA (rDNA) is comprised of deoxyribonucleic acid (DNA) fragments and longer length molecules originating from the host organism that may be present in samples from recombinant biological processes. Although similar in basic structural base pair units, rDNA may exist in different sizes and physical forms. Interest in measuring rDNA in recombinant products is based primarily on demonstration of effective purification during manufacturing, but also on some hypothetical concerns that, in rare cases, depending on the host expression system, some DNA sequences may be potentially infectious or oncogenic (e.g., HIV virus and the Ras oncogene, respectively). Recent studies suggest that a sequence known as long interspersed nucleotide element-1 (LINE-1), widely distributed in the mammalian genome, is active as a retrotransposon that can be transcribed to RNA, reverse-transcribed into DNA and inserts into a new site in genome. This integration process could potentially disrupt critical gene functions or induce tumorigenesis in mammals. Genomic DNA from microbial sources, on the other hand, could add to risk of immunogenicity to the target recombinant protein being expressed, due to the high CpG content and unmethylated DNA sequence. For these and other reasons, it is necessary for manufacturers to show clearance of DNA throughout production processes and to confirm low levels in the final drug substance using an appropriately specific and quantitative analytical method. The heterogeneity of potential rDNA sequences that might be makes the testing of all potential analytes challenging. The most common methodology for rDNA quantitation used currently is real-time polymerase chain reaction (RT-PCR), a robust and proven technology. Like most rDNA quantitation methods, the specificity of RT-PCR is limited by the sequences to which the primers are directed. To address this, primase-based whole genome amplification is introduced herein. This paper will review the recent advancement in rDNA quantitation and recent findings regarding potential risks of immunogenicity, infectivity, and oncogenicity of rDNA.

Benfotiamine increases glucose oxidation and downregulates NADPH oxidase 4 expression in cultured human myotubes exposed to both normal and high glucose concentrations

The aim of the present work was to study the effects of benfotiamine (S-benzoylthiamine O-monophosphate) on glucose and lipid metabolism and gene expression in differentiated human skeletal muscle cells (myotubes) incubated for 4days under normal (5.5mM glucose) and hyperglycemic (20mM glucose) conditions. Myotubes established from lean, healthy volunteers were treated with benfotiamine for 4days. Glucose and lipid metabolism were studied with labeled precursors. Gene expression was measured using real-time polymerase chain reaction (qPCR) and microarray technology. Benfotiamine significantly increased glucose oxidation under normoglycemic (35 and 49% increase at 100 and 200μM benfotiamine, respectively) as well as hyperglycemic conditions (70% increase at 200μM benfotiamine). Benfotiamine also increased glucose uptake. In comparison, thiamine (200μM) increased overall glucose metabolism but did not change glucose oxidation. In contrast to glucose, mitochondrial lipid oxidation and overall lipid metabolism were unchanged by benfotiamine. The expression of NADPH oxidase 4 (NOX4) was significantly downregulated by benfotiamine treatment under both normo- and hyperglycemic conditions. Gene set enrichment analysis (GSEA) showed that befotiamine increased peroxisomal lipid oxidation and organelle (mitochondrial) membrane function. In conclusion, benfotiamine increases mitochondrial glucose oxidation in myotubes and downregulates NOX4 expression. These findings may be of relevance to type 2 diabetes where reversal of reduced glucose oxidation and mitochondrial capacity is a desirable goal.

Mitochondrial Gene Expression in Directly Irradiated and Nonirradiated Bystander Cells

Nontargeted cellular effects by ionizing radiation are well documented. The bystander effect is a nontargeted phenomenon wherein the irradiated cells communicate and induce changes in nonirradiated cells. The nature of the bystander signal and how it impacts unirradiated cells remain to be elucidated. Examination of molecular changes could lead to the identification of molecular pathways underlying the bystander effect. In this study, mitochondrial gene transcriptional changes in bystander cells were monitored to gain insight into the participation of mitochondria in this response. The modulation of mitochondrial gene expression in medium-exchanged bystander cells was determined in human lymphoblast TK6 cells by employing the real-time polymerase chain reaction technology. The examination of the relative expression of mitochondrial genes involved in various metabolic functions indicated that MT-ND1, MT-ND5, and MT-ND6 encoding NADH dehydrogenases were upregulated in directly irradiated cells but repressed in bystander cells. The differences in the expression levels were statistically significant among irradiated and bystander cells. The adenosine triphosphate (ATP) synthases MT-ATP6 and MT-ATP8 were upregulated in both irradiated and bystander cells. These results point to the involvement of mitochondrial gene modulation in directly irradiated and bystander cells and provide evidence that mitochondrial gene expression response is part of a complex stress response operating in radiation-treated cells.

Association of PTPN22 gene polymorphism and systemic lupus erythematosus in a cohort of Egyptian patients: impact on clinical and laboratory results

To assess the possible association between the protein tyrosine phosphatases non-receptor 22 (PTPN22) gene 1858 CT polymorphism and the predisposition to systemic lupus erythematosus (SLE) in Egyptian patients and its influence on clinical and laboratory parameters. PTPN22 gene 1858 CT polymorphisms were analyzed in forty SLE patients and 20 normal controls by real-time polymerase chain reaction (PCR) technology, using the TaqMan 5-allele discrimination assay. Detailed history, clinical examination, and investigations were done to detect various organ involvement. The homozygous genotype TT was absent in both SLE and controls. The CC genotype was observed in 47.5% SLE and 80% controls; the CT genotype was found in 52.5% patients and 20% controls. The frequencies of the C and T alleles were 74 and 26% in SLE and 90 and 10% in controls, respectively. The presence of CT genotype increased the risk for developing SLE by 4.42. Renal involvement was significantly higher in SLE patients with CT (76.2%) compared to those with CC genotype (42.1%).

Real‐Time Quantitative Polymerase Chain Reaction Analysis of Patients With Refractory Chronic Periodontitis

Periodontitis is a complex multifactorial disease and is typically polygenic in origin. Genes play a fundamental part in each biologic process forming complex networks of interactions. However, only some genes have a high number of interactions with other genes in the network and may, therefore, be considered to play an important role. In a preliminary bioinformatic analysis, five genes that showed a higher number of interactions were identified and termed leader genes. In the present study, we use real-time quantitative polymerase chain reaction (PCR) technology to evaluate the expression levels of leader genes in the leukocytes of 10 patients with refractory chronic periodontitis and compare the expression levels with those of the same genes in 24 healthy patients. Blood was collected from 24 healthy human subjects and 10 patients with refractory chronic periodontitis and placed into heparinized blood collection tubes by personnel trained in phlebotomy using a sterile technique. Blood leukocyte cells were immediately lysed by using a kit for total RNA purification from human whole blood. Complementary DNA (cDNA) synthesis was obtained from total RNA and then real-time quantitative PCR was performed. PCR efficiencies were calculated with a relative standard curve derived from a five cDNA dilution series in triplicate that gave regression coefficients >0.98 and efficiencies >96%. The standard curves were obtained using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and growth factor receptor binding protein 2 (GRB2), casitas B-lineage lymphoma (CBL), nuclear factor-KB1 (NFKB1), and REL-A (gene for transcription factor p65) gene primers and amplified with 1.6, 8, 40, 200, and 1,000 ng/μL total cDNA. Curves obtained for each sample showed a linear relationship between RNA concentrations and the cycle threshold value of real-time quantitative PCR for all genes. Data were expressed as mean ± SE (SEM). The groups were compared to the analysis of variance. A probability value <0.01 was considered statistically significant. The present study agrees with the preliminary bioinformatics analysis. In our experiments, the association of pathology with the genes was statistically significant for GRB2 and CBL (P <0.01), and it was not statistically significant for REL-A and NFKB1. This article lends support to our preliminary hypothesis that assigned an important role in refractory aggressive periodontitis to leader genes.

The variability of soil microbial community composition of different types of tidal wetland in Chongming Dongtan and its effect on soil microbial respiration

Previous studies have shown that the soil enzyme activity and microbial respiration intensities varied in two different types of tidal wetland in Chongming Dongtan, the first a sandy soil in a scouring bank with Phragmites australis and the second a saline-alkali clay soil in silting bank with P. australis/ Spartina alterniflora/ Scirpus mariqueter, resulting in different organic carbon reservation capabilities; however, their microbial biomass did not differ significantly. To clarify the microbial mechanism that explains the variability of soil respiration among different wetland areas, the community structure and abundance of soil microorganisms in different types of wetland were investigated using denaturing gradient gel electrophoresis (DGGE) plus real-time quantitative polymerase chain reaction (PCR) technologies, and the relationship between soil environmental factors and the microbial community structure and the soil respiration intensity was elucidated. The results revealed that the soil microbial diversity and community structure differed between the two typical wetland areas. The common population was uncultured bacterium in both areas, and the most abundant community was α-, β-, γ- Proteobacteria, which play an important role in the cycling of carbon in soil. However, the abundance of α- Proteobacteria in Area A was 18.2% of that in Area B ( P <0.05), while the β- Proteobacteria in Area A was 3.23 times higher than that in Area B ( P <0.05). In addition, one cellulose-degrading bacteria, uncultured Bacilli, was detected in Area A. PCA (Principal component analysis) revealed that γ- Proteobacteria and β- Proteobacteria had the greatest impact on soil respiration intensity. Both soil water content and salinity depressed the propagation of β- Proteobacteria. Considering the similar microbial biomass and abundance of γ- Proteobacteria between the two areas, the lower level of β- Proteobacteria, uncultured Bacilli bacterium in Area B might be important factors involved in the lower soil respiration, and hence the higher soil organic carbon reservation capability in Area B.

Genomic signatures of cancer: Basis for individualized risk assessment, selective staging and therapy

The development of DNA microarray and quantitative real-time polymerase chain reaction technologies has allowed for precise quantitation of RNA expression of hundreds to thousands of genes. These technologies have significantly impacted the study and understanding of cancer in terms of its molecular and genetic characteristics. In this review article, breast cancer, colon cancer, and adrenal carcinoma have been chosen to illustrate the principle and techniques of genomic profiling and to illustrate how such methods may be used to develop genomic signatures for personalized risk assessment and to individualize patient treatment.

Sensitive quantitation of minimal residual disease in chronic myeloid leukemia using nanofluidic digital polymerase chain reaction assay

Undetectable BCR–ABL transcripts in patients with chronic myeloid leukemia (CML) should not be regarded as indicative of a cure, due to the sensitivity limit of current real-time quantitative polymerase chain reaction (RQ-PCR) technology. To demonstrate the feasibility of more sensitive approaches, 62 samples from 43 patients with CML were screened by conventional RQ-PCR, replicate RQ-PCR (rRQ-PCR), and/or nanofluidic digital PCR (dPCR). First, we confirmed the correlation of dPCR to conventional RQ-PCR using 30 patient samples with various minimal residual disease (MRD) levels. When the sensitivity limits were determined using cell line and patient sample dilutions, rRQ-PCR and dPCR with pre-amplification showed 2–3 log improvement compared to conventional RQ-PCR, and 24 of 32 PCR negative samples as assayed by conventional RQ-PCR showed detectable BCR–ABL in rRQ-PCR and/or dPCR. More important, using dPCR in conjunction with a pre-amplification step, a continuous decline in MRD level could be precisely monitored even after it became undetectable by conventional RQ-PCR. In this study, both rRQ-PCR and dPCR demonstrated successful detection of BCR–ABL transcripts not detectable in conventional RQ-PCR, and these data show the potential feasibility of highly sensitive PCR approaches for molecular monitoring and clinical relevance in future CML management by allowing further characterization of patients who achieve PCR negativity in a conventional RQ-PCR assay.