Exponential Phase Of Amplification Research Articles

Real-Time PCR as a Tool to Study Weed Biology

Real-time polymerase chain reaction (real-time PCR), also known as quantitative PCR, is used to determine relative gene expression or to quantify exact levels of mRNA in cells or tissues. Before the advent of real-time PCR, the major difficulty associated with traditional quantitative or semiquantitative PCR was to ensure that PCR reactions were quantified within the exponential phase of amplification. Real-time PCR alleviates that problem by detecting and quantifying fluorescent signals after each amplification cycle. Additionally, it does not require running gels and thus is able to produce data in 2 to 3 h. Four different types of chemistries, DNA-binding agents (SYBR Green), hydrolysis probes (TaqMan), hairpin probes (molecular beacons, scorpions), and fluorescent-labeled hybridization probes (Light Cycler), have been commonly used for real-time PCR. Among those chemistries, SYBR Green is the most economical choice. We have used real-time PCR and SYBR Green to examine the expression of a number of leafy spurge genes after growth induction and during normal seasonal growth. Because no reliable endogenous reference genes have been identified in leafy spurge, we performed PCR without an endogenous reference gene and analyzed messenger RNA (mRNA) expression based on the threshold cycle (CT) value of amplification. Excluding an endogenous reference gene from that data analysis was rather straightforward and reliable if RNA was properly prepared and quantified. Given that genomic tools, such as expressed sequence tags (ESTs), and their expression profiles are lacking for most weedy species, avoiding the use of endogenous reference genes in real-time PCR simplifies the optimization process and reduces the cost tremendously. However, we found that using a passive reference dye (ROX) to normalize non-PCR–related fluctuations in fluorescent signal is desirable.

Increased CUG Triplet Repeat-binding Protein-1 Predisposes to Impaired Adipogenesis with Aging

Preadipocyte differentiation capacity declines between middle and old age. Expression of the adipogenic transcription factors, CCAAT/enhancer-binding protein (C/EBP) alpha and peroxisome proliferator-activated receptor gamma (PPARgamma), is lower in differentiating preadipocytes from old than young animals, although no age-related changes occur in C/EBPbeta mRNA, which is upstream of C/EBPalpha and PPARgamma. C/EBPbeta-liver-enriched inhibitory protein (C/EBPbeta-LIP), a truncated C/EBPbeta isoform that is a dominant inhibitor of differentiation, increases with aging in rat fat tissue and preadipocytes. CUG triplet repeat-binding protein-1 (CUGBP1) binds to C/EBPbeta mRNA, increasing C/EBPbeta-LIP translation. Abundance and nucleotide binding activity of CUGBP1 increased with aging in preadipocytes. CUGBP1 overexpression in preadipocytes from young animals increased C/EBPbeta-LIP and impaired adipogenesis. Decreasing CUGBP1 in preadipocytes from old rats by RNA interference reduced C/EBPbeta-LIP abundance and promoted adipogenesis. Tumor necrosis factor-alpha, levels of which are elevated in fat tissue with aging, increased CUGBP1 protein, CUGBP1 binding activity, and C/EBPbeta-LIP in preadipocytes from young rats. Thus, CUGBP1 contributes to regulation of adipogenesis in primary preadipocytes and is responsive to tumor necrosis factor-alpha. With aging, preadipocyte CUGBP1 abundance and activity increases, resulting in enhanced translation of the C/EBPbeta-LIP isoform, thereby blocking effects of adipogenic transcription factors, predisposing preadipocytes from old animals to resist adipogenesis. Altered translational processing, possibly related to changes in cytokine milieu and activation of stress responses, may contribute to changes in progenitor differentiation and tissue function with aging.

Targeted Overexpression of Sarcolipin in the Mouse Heart Decreases Sarcoplasmic Reticulum Calcium Transport and Cardiac Contractility

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca(2+) ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by beta-adrenergic agonists.

Identification of Deletion Carriers in Duchenne/Becker Muscular Dystrophy Families Using a Digoxigenin-Labeled Quantitative Polymerase Chain Reaction Technique.

Background: A quantitative polymerase chain reaction (PCR) technique based on the incorporation of digoxigenin (DIG), and visualization of the labeled fragments for the detection of deletion carriers in Duchenne/Becker muscular dystrophy families has been developed. Methods and Results: Sixty-five DNA samples taken from mothers and/or sisters of familial and sporadic deletion patients were investigated in the exponential phase of amplification. All obligate carriers were correctly identified using this technique. In more than 95% of deletion families, possible carriers could be screened by using four different multiplex systems specifically designed to increase the efficiency of the detection. Deletions were found to be present in 42% of possible carriers. All these results were confirmed by computer-assisted laser densitometry. Conclusions: Dosage analysis by DIG-labeled quantitative PCR is a reliable and accurate technique for detecting Duchenne/Becker muscular dystrophy deletion carriers.

Multiplex Fluorescent RT-PCR to Quantify Leukemic Fusion Transcripts

The detection of chimeric transcripts derived from aberrant chromosomal fusion events provides an exceptionally valuable toolfor the diagnosis of leukemia. We have developed a simple, inexpensive, reproducible, and automated method to quantify RT-PCR products. Our approach utilizesfluorescent PCRfor the co-ampification of the specific fusion transcript with an internal control (HPRT). We have also combined the advantages of real-time quantitative PCR, namely continuous fluorescent detection of PCR products with the low cost of an endpoint assay by examining in a novel manner the amount offluorescent PCR product generated during the exponential phase of amplification. This has been achieved by using the automated loading and quantification capacity of a laser-induced fluorescence capillary electrophoresis system, the ABI PRIsMS 310A, so that we can effectively monitor amplification during the exponential phase cheaply, reproducibly, and in a sensitive manner. We have carefully verified our new technique using five leukemia cell lines, each expressing a differentfusion transcript. Specificity and reproducibility (cy within 10%) have been examined and demonstrate the excellent precision of our technology. The high sensitivity levels of at least 10(-4) to 10(-6) obtainedfor the serial dilutions of the five cell lines validate the choice of our fluorescent PCR as a comparable method to other more complicated and expensive methods. Our results have allowed us to quantify PCR products and the amount of chimeric mRNA originating from the translocation breakpoint. We demonstrate that our novelfluorescent method is useful to detect and quantify residual leukemic cells in patients undergoing therapy.

Ribosomal 18S RNA Prevails over Glyceraldehyde-3-Phosphate Dehydrogenase and β-Actin Genes as Internal Standard for Quantitative Comparison of mRNA Levels in Invasive and Noninvasive Human Melanoma Cell Subpopulations

The comparison of the gene expression profiles between two subpopulations of melanoma cells (1C8 and T1C3) derived from the tumor of one patient by cDNA array revealed differences in GAPDH and β-actin gene levels. These two housekeeping genes were up-regulated in invasive T1C3 melanoma cells compared to noninvasive 1C8 cells. Since cDNA array results were not confirmed by conventional RT-PCR throughout the exponential phase of amplification, we performed duplex relative RT-PCR using ribosomal 18S RNA as internal standard including competimer technology. Statistical analyses provided significant evidence that invasive T1C3 melanoma cells exhibited a twofold higher mRNA level of both GAPDH and β-actin than noninvasive 1C8 cells. This study demonstrates that the duplex relative RT-PCR procedure including ribosomal 18S RNA as internal standard and competimer technology is precise for RNA quantification and is tailored for cDNA array validation. Our data provide molecular evidence that cellular subpopulations of the same pathological origin are highly heterogeneous and extend the concept that the selection of an appropriate internal control for comparative mRNA analysis should be adapted to each model of human cancers.

Real-time polymerase chain reaction assay for cell-associated HTLV type I DNA viral load.

We have developed a quantitative real-time PCR assay for HTLV-I DNA. This assay approach uses real-time monitoring of fluorescent signal generation as a consequence of Taq-mediated amplification of specific target sequences to allow real-time kinetic analysis of amplicon production. This kinetic approach yields excellent sensitivity and an extremely broad linear dynamic range, and ensures that quantitation is based on analysis during the exponential phase of amplification, regardless of the input template copy number. The HTLV-I DNA assay has a nominal threshold sensitivity of 10 copy Eq/reaction, although single-copy plasmid template can be detected at frequencies consistent with statistical prediction. The linear dynamic range is in excess of 5 logs. Interassay reproducibility averages 14% (coefficient of variation) for control templates over a range of 10(1) to 10(6) copy Eq/reaction and 25%, based on studies of extraction and analysis of replicate aliquots of PBMC specimens from HTLV-I-infected subjects. The primer/probe combination targets tax sequences conserved across described HTLV-I and HTLV-II isolates. Parallel quantitation in the same samples of an endogenous sequence present at a known copy number per cell allows normalization of results for potential variation in DNA recovery. Availability of this assay should facilitate studies of basic pathogenesis and clinical evaluation of HTLV-I and HTLV-II infection, as well as assessment of therapeutic approaches.

Relative quantitative RT-PCR protocol for TrkB expression in neuroblastoma using GAPD as an internal control.

An RT-PCR protocol for the relative quantitative measurement of TrkB transcripts using glyceraldehyde-3-phosphate-dehydrogenase (GAPD) transcripts as an internal control is described. Both TrkB and GAPD sequences were co-amplified in the exponential phase of amplification using 5'-biotinylated primers. The PCR products were subjected to PAGE, electro-transferred to nylon membrane and detected by a chemiluminescent procedure using alkaline phosphatase conjugated with avidin. Signals detected on X-ray film were analyzed by densitometry. The ratio between TrkB and GAPD expression levels was determined to normalize the expression levels of TrkB transcripts. Initially, strong signals of GAPD transcripts led to overexposure of X-ray film compared to those of TrkB, which causes difficulties in the accurate determination of the TrkB/GAPD ratio. To circumvent this problem, uniformly biotinylated GAPD primers were replaced with a mixture of biotinylated and non-biotinylated GAPD primers of the same sequence and concentration. GAPD signals detected on X-ray film were proportionally decreased as the amount of biotin-labeled primers was reduced in the total GAPD primers. This modification enabled both GAPD and TrkB signals to be analyzed within the linear range of X-ray film detection without affecting the amplification efficiency of TrkB sequence. Use of composite primers may have a wide range of applicability in quantitative analysis of nucleic acids.

Gender- and androgen-related influence on the expression of proto-oncogene and apoptotic factor mRNAS in lacrimal glands of autoimmune and non-autoimmune mice

Our previous studies have shown that the mRNA levels of c-myb, c-myc, bcl-2 and p53 are higher, and partial Fas antigen (i.e. exons 1–2) lower, in lacrimal tissues of female, as compared to male, MRL/lpr mice, which are a model of Sjögren's syndrome. We have also found that this gender-related difference in bcl-2 and c-myb expression appears to be due to the influence of androgens. To extend these findings, we sought to determine: first, whether these gender- and/or hormone-associated variations in mRNA content are unique to MRL/lpr mice, or are also present in lacrimal glands of other murine strains, including autoimmune NZB/NZW F1 (F1) and non-obese diabetic (NOD), as well as non-autoimmune C3H/HeJ (C3H) and BALB/c, mice; and second, whether the levels of these apoptotic factor mRNAs are altered in lacrimal tissues of mice (i.e. testicular feminized (Tfm) with dysfunctional androgen receptors, as compared to glandular amounts in their ‘normal’ controls (i.e. Tabby). Lacrimal tissues were obtained from adult mice, which were either untreated or treated with placebo or testosterone for 21 days. Glands were processed for the analysis of proto-oncogene mRNAs by RT-PCR (at exponential phase of amplification) and data were standardized to the corresponding levels of β-actin mRNA. Our results demonstrate that Fas antigen, Fas ligand, c-myb, c-myc, bcl-2, Bax and p53 mRNAs are present in lacrimal tissues of F1, NOD, C3H, BALB/c, Tabby and Tfm mice. The relative levels of Fas antigen mRNA are consistently higher in glands of males, whereas amounts of bcl-2 mRNA are greater in tissues of F1, C3H and BALB/c females. Testosterone administration induced a significant increase in the lacrimal gland content of Bax mRNA, but a striking decrease in the lacrimal tissue level of bcl-2 mRNA in F1 and C3H mice. Lacrimal glands of Tfm mice contained elevated amounts of bcl-2 mRNA, as compared to values in tissues of their Tabby controls. In summary, our findings show that fundamental gender-related differences exist in the expression of genes associated with programmed cell death in lacrimal glands of autoimmune and normal mice. In addition, some of these differences may be due, at least in part, to the effect of androgens.

Plasma SIV RNA viral load determination by real-time quantification of product generation in reverse transcriptase-polymerase chain reaction.

Internally controlled RT-PCR methods (QC-RT-PCR) for quantification of SIV RNA are effective, but are relatively cumbersome, expensive, and time and labor intensive. For greater throughput and efficiency, we have developed a method for quantification of plasma SIV RNA levels by real-time RT-PCR using the Applied Biosystems Prism 7700 sequence detection system. This assay format allows real-time kinetic analysis of PCR product generation, providing a broad linear dynamic range and ensuring that quantification is based on analysis during the exponential phase of amplification, regardless of the input template copy number. Simultaneous amplification and analysis eliminates any requirement for handling amplified products, increasing throughput and eliminating a potential source of assay contamination. The assay we have developed for quantification of SIV RNA has a nominal threshold sensitivity of 300 copy Eq/ml of plasma, although as little as 10 copy Eq/reaction of SIV RNA template can be detected. The linear dynamic range is in excess of 5 logs. Interassay reproducibility averages 25% (coefficient of variation), based on studies of extraction and analysis of replicate aliquots of the same plasma specimens. The combination of sensitivity, precision, and broad dynamic range allows reliable quantification of viral load even during dynamic phases of SIV infection, such as through the onset and resolution of primary infection, or during treatment with antiretroviral agents. The primer-probe combinations we have developed allow quantification of SIV isolates most commonly used for experimental studies. Availability of this assay should greatly facilitate studies of basic pathogenesis and evaluation of therapeutic and prophylactic approaches in the SIV-infected macaque.

Quantitative reverse transcriptase-PCR amplification of cytokine mRNA in liver biopsy specimens using a non-competitive method.

Reverse transcriptase-PCR (RT-PCR) amplification of mRNA is often the only technique able to detect expression of cytokine mRNA in small samples. The aim of this work was to investigate the utility of a non-competitive RT-PCR which used external standards to quantitate TNF-alpha mRNA in liver biopsy specimens from liver transplant patients. It involved removal of aliquots from the PCR reaction at successive cycles, followed by dot-blotting of the samples onto nylon membrane and hybridization with a radioactively-labelled internal probe. Phosphorimage analysis of the labelled membranes allowed quantitation of the relative amount of PCR product at successive cycles. Plots of log(counts) versus cycle number showed straight lines in the exponential phase of amplification. The slopes of these lines showed the efficiency of amplification, which ranged from 76 to 87% for liver biopsy samples. Estimation of liver biopsy levels of TNF-alpha in two separate PCR amplifications showed low inter-assay variability (r2 = 0.98). Comparison of two separate cDNA syntheses also showed good correlation (r2 = 0.81, P < 0.0001), although not as good as for the PCR alone. This shows that variation in efficiency of cDNA synthesis is likely to contribute as much or more to variability of the analysis as variations in PCR amplification.

Quantitative analysis of 16S rDNA using competitive PCR and the QPCR System 5000.

A method for precise and accurate quantification of 16S rDNA has been developed that uses competitive PCR and the QPCR System 5000. The method is based on co-amplification of 16S rDNA sequences, along with an internal standard sequence, using only one set of conserved eubacterial primers. Co-amplified PCR products are rapidly identified and quantified by measuring the electrochemiluminescent signals from specific oligonucleotide reporter probes that are directed against a hypervariable 16S rDNA sequence. Because in the exponential phase of amplification the different target sequences and the internal standard sequence are amplified with the same efficiency, unknown amounts of a target sequence in a sample can be inferred by extrapolating against a standard curve that is generated for the internal standard sequence. This method provides a rapid, nonradioactive and reliable way to simultaneously quantify different specific 16S rDNA targets that are present in low numbers, and may thus be suitable for enumeration of specific target microorganisms in environmental samples.

Cytokine gene expression in rejecting and tolerant rat lung allograft models: analysis by RT-PCR

Cytokine gene expression is a critical component of the lung allograft rejection (AR) response and tolerance development in rat models. In order to determine the specificity of cytokine gene expression for AR and tolerance, we examined cytokine (interleukin-2) (IL-2), (gamma-interferon) (γ-IFN), IL-4, IL-10 and tumor necrosis factor-α (TNF-α) and control (cyclophilin) mRNA levels in two models of rat lung allograft rejection by RT-PCR (reverse transcriptase polymerase chain reaction), Southern blotting. The first model (WKY→F344) develops a mild to moderate lymphocytic infiltrate on days 14–21 post-transplant (stage II–III AR), which spontaneously resolves by day 35 post-transplant with subsequent development of allograft tolerance (grafts surviving without evidence of AR for > 140 days). Conversely, F344→WKY develops a similar lymphocytic infiltrate by day 14 post-transplant, but by day 21 post-transplant the graft shows severe AR (stage III–IV) and has haemorrhagic infarction with alveolar haemorrhage. Methods: RNA was extracted from allografts removed on days 3, 7, 13, 21, 35 and 42 post-transplant. Five animals for each group (WKY→F344) and F344→WKY) were examined at each time point, except that no animals in the F344→WKY were examined on day 42. cDNA was synthesized from total extracted RNA and primers specific for rat TNF-α, rat IL-2, rat γ-IFN, rat IL-4, rat-IL-10 and rat cyclophilin were used for gene-specific amplification. (TNF-α, γ-IFN, IL-10, 20 cycles; IL-2, IL-4, 30 cycles; cyclophilin, 20 cycles). The cycles numbers chosen for comparison were found to be optimal during preliminary experiments and occurred during the exponential phase of amplification. PCR products were electrophoresed on a polyacrylamide gel and silver-stained. Gels were subsequently electrotransferred to nylon membranes which were probed with murine cDNAs specific for IL-2, γ-IFN IL-4, IL-10 and TNF-γ. Results: Cyclophilin gene expression was similar for both models at all time points tested; this also served as an internal standard for RT-PCR. In the WKY→F344 tolerance model, TNF-α mRNA levels were not detectable on days 3 and 7 post-transplant, were at very low levels on day 14 and were undetectable on day 21 post-transplant. In marked contrast, the F344→WKY rejection model showed TNF-α mRNA present on day 3 which increased markedly on day 7 and peaked on day 14 post-transplant. TNF-α mRNA levels decreased on days 21 and 35 post-transplant, a time when the lung was undergoing AR. The pattern of IL-2 and γ-IFN mRNA expression was similar to that for TNF-α. However, IL-2 mRNA was clearly detectable in the WKY→F344 tolerance model on day 7 and γ-IFN was not present until day 14 post-transplant. The F344→WKY rejection model showed very high levels of IL-2 and γ-IFN on day 3 which peaked on day 14. The ratio of IL-2/IL 10 in the F344→WKY rejection model was more than five times that seen in the WKY→F344 tolerance model on day 3 ( p < 0.0005). The ratio of IL-2/IL-4 was higher (1.5 times) in the F344→WKY rejection model than in the WKY→F344 tolerance model ( p < 0.007) on day 3. On day 14 post-transplant, the IL-2/IL-10 ratio in the F344→WKY rejection model was three times that of the tolerance model ( p < 0.0015). The IL-2/IL-4 ratio was 3.5 times greater in the WKY→F344 tolerance model than in the rejection model ( p < 0.003). This was due to equal expression of IL-2 and IL-4 in the rejection model, but poor IL-4 expression in the tolerance model. Conclusions: 1) The WKY→F344 tolerance model develops mild to moderate lymphocytic infiltrates on day 14 which is associated with low level IL-2, γ-IFN and TNF-α gene expression. IL-10 and IL-4 are present at day 3; however, by day 14, IL-10 is the predominantly expressed Th2 cytokine and IL-4 is not expressed. The infiltrates ultimately resolve and the animals develop a functional tolerance to their grafts. 2) The F344→WKY rejection model shows similar lymphocytic infiltrates on day 14, but diverges to a severe rejection which is associated with very early and high levels of IL-2, IL-4, γ-IFN and TNF-α mRNA expression. IL-10 is not expressed on day 3, and only poorly expressed on day 14. 3) These data suggest that delayed and low level expression of cytokines can be seen in animals developing tolerance to their grafts while early and abundant expression of cytokines (IL-2, IL-4, γ-IFN and TNF-α) is associated with AR and ultimate graft loss. It also appears that, in this system, IL-10 expression is critical for development of tolerance while IL-4 expression occurs early in both the rejection and tolerant models, but it expressed poorly in the tolerant models on day 14.

Analysis of quantitative PCR for the diagnosis of deletion and duplication carriers in the dystrophin gene.

A direct, non-radioactive method of quantitative PCR amplification has been investigated for the diagnosis of deletion and duplication carriers in the dystrophin gene. The simultaneous amplification of two loci, or several loci using multiplex PCR, allows for the direct comparison of relative amounts of products from normal homozygous loci and potentially heterozygous deleted/duplicated loci. Sufficient cycles of PCR are performed to enable visual analysis or densitometric quantification of products on ethidium bromide stained gels. The method has been verified in blind trials performed on known genotypes and by showing that under the conditions used the assay remains within the exponential phase of amplification.