Real-time PCR Machine Research Articles

Selenoprotein H Is a Redox-sensing High Mobility Group Family DNA-binding Protein That Up-regulates Genes Involved in Glutathione Synthesis and Phase II Detoxification

Selenoprotein H is a recently identified member of the selenoprotein family whose function is not fully known. Previous studies from our laboratory and others showed that Drosophila melanogaster selenoprotein H is essential for viability and antioxidant defense. In this study we investigated the function of human selenoprotein H in murine hippocampal HT22 cells engineered to stably overexpress the protein. After treatment of cells with L-buthionine-(S,R)-sulfoximine to deplete glutathione, selenoprotein H-overexpressing cells exhibited higher levels of total glutathione, total antioxidant capacities, and glutathione peroxidase enzymatic activity than did vector control cells. Overexpression of selenoprotein H also up-regulated the mRNA levels of endogenous selenoprotein H, glutamylcysteine synthetase heavy and light chains, and glutathione S-transferases Alpha 2, Alpha 4, and Omega 1. The amino acid sequence of selenoprotein H contains four putative nuclear localization sequences and an AT-hook motif, a small DNA-binding domain first identified in high mobility group proteins. Chromatin immunoprecipitation using a green fluorescent protein-selenoprotein H fusion revealed binding to sequences containing heat shock and/or stress response elements. Thus, selenoprotein H is a redox-responsive DNA-binding protein of the AT-hook family and functions in regulating expression levels of genes involved in de novo glutathione synthesis and phase II detoxification in response to redox status.

Transient Receptor Potential Vanilloid 1 Mediates Hyperalgesia and Is Up-Regulated in Rats With Chronic Pancreatitis

The neurobiologic basis of pancreatic hyperalgesia in chronic pancreatitis (CP) is understood poorly and there is a need to identify novel therapeutic targets. Our aim was to study the role of the transient receptor potential vanilloid 1 (TRPV1), a key integrator of noxious stimuli, in the pathogenesis of pancreatic pain in a rat model of CP. CP was induced in rats by intraductal injection of trinitrobenzene sulfonic acid. TRPV1 currents in pancreas-specific DRG neurons were measured using perforated patch-clamp techniques. Reverse-transcription polymerase chain reaction was used to measure mRNA expression of TRPV1 in these neurons after laser capture microdissection. Immunofluorescence and Western blot analysis, using TRPV1-specific antibodies, also were performed. Pancreatic hyperalgesia was assessed by rat's nocifensive behavior to electrical stimulation of the pancreas. CP was associated with a 4-fold increase in capsaicin-induced current density (P < .02), along with an increase in the proportion of pancreas-specific DRG neurons that responded to capsaicin (52.9% in controls vs 79.0% in CP; P < .05). CP also was associated with a significant increase in TRPV1 expression both at the messenger RNA and protein level in whole thoracic DRGs and pancreas-specific sensory neurons. Systemic administration of the TRPV1 antagonist SB-366791 markedly reduced both visceral pain behavior and referred somatic hyperalgesia in rats with CP, but not in control animals. TRPV1 up-regulation and sensitization is a specific molecular mechanism contributing to hyperalgesia in CP and represents a useful target for treating pancreatic hyperalgesia caused by inflammation.

Rapid detection of Escherichia coli virulence factor genes using multiplex real-time TaqMan ® PCR assays

Three multiplex real-time TaqMan ® PCR assays were developed for the detection of Escherichia coli virulence factor genes in veterinary samples. Target virulence factors chosen were the fimbriae K88 (F4), K99 (F5), F41, F17, F18 and 987p (F6) and the toxins LT, STa and CDT IV. Detection of genes coding GAD were included in each assay as an internal control. These assays allow rapid identification of virulence factor genes using identical cycling conditions on an Mx3000P™ real-time PCR machine with the capacity to test up to 20 strains for 9 virulence genes in 1 h.

Tumor Necrosis Factor α Induces Spermidine/Spermine N1-Acetyltransferase through Nuclear Factor κBin Non-small Cell Lung Cancer Cells

Tumor necrosis factor alpha (TNFalpha) is a potent pleiotropic cytokine produced by many cells in response to inflammatory stress. The molecular mechanisms responsible for the multiple biological activities of TNFalpha are due to its ability to activate multiple signal transduction pathways, including nuclear factor kappaB (NFkappaB), which plays critical roles in cell proliferation and survival. TNFalpha displays both apoptotic and antiapoptotic properties, depending on the nature of the stimulus and the activation status of certain signaling pathways. Here we show that TNFalpha can lead to the induction of NFkappaB signaling with a concomitant increase in spermidine/spermine N(1)-acetyltransferase (SSAT) expression in A549 and H157 non-small cell lung cancer cells. Induction of SSAT, a stress-inducible gene that encodes a rate-limiting polyamine catabolic enzyme, leads to lower intracellular polyamine contents and has been associated with decreased cell growth and increased apoptosis. Stable overexpression of a mutant, dominant negative IkappaBalpha protein led to the suppression of SSAT induction by TNFalpha in these cells, thereby substantiating a role of NFkappaB in the induction of SSAT by TNFalpha. SSAT promoter deletion constructs led to the identification of three potential NFkappaB response elements in the SSAT gene. Electromobility shift assays, chromatin immunoprecipitation experiments and mutational studies confirmed that two of the three NFkappaB response elements play an important role in the regulation of SSAT in response to TNFalpha. The results of these studies indicate that a common mediator of inflammation can lead to the induction of SSAT expression by activating the NFkappaB signaling pathway in non-small cell lung cancer cells.

The Alternative Stimulatory G Protein α-Subunit XLαs Is a Critical Regulator of Energy and Glucose Metabolism and Sympathetic Nerve Activity in Adult Mice

The complex imprinted Gnas locus encodes several gene products including G(s)alpha, the ubiquitously expressed G protein alpha-subunit required for receptor-stimulated cAMP generation, and the neuroendocrine-specific G(s)alpha isoform XLalphas. XLalphas is only expressed from the paternal allele, whereas G(s)alpha is biallelically expressed in most tissues. XLalphas knock-out mice (Gnasxl(m+/p-)) have poor suckling and perinatal lethality, implicating XLalphas as critical for postnatal feeding. We have now examined the metabolic phenotype of adult Gnasxl(m+/p-) mice. Gnasxl(m+/p-) mice had reduced fat mass and lipid accumulation in adipose tissue, with increased food intake and metabolic rates. Gene expression profiling was consistent with increased lipid metabolism in adipose tissue. These changes likely result from increased sympathetic nervous system activity rather than adipose cell-autonomous effects, as we found that XLalphas is not normally expressed in adult adipose tissue, and Gnasxl(m+/p-) mice had increased urinary norepinephrine levels but not increased metabolic responsiveness to a beta3-adrenergic agonist. Gnasxl(m+/p-) mice were hypolipidemic and had increased glucose tolerance and insulin sensitivity. The similar metabolic profile observed in some prior paternal Gnas knock-out models results from XLalphas deficiency (or deficiency of the related alternative truncated protein XLN1). XLalphas (or XLN1) is a negative regulator of sympathetic nervous system activity in mice.

MOLECULAR COMPUTATION: A DNA‐BASED MODEL FOR SOLVING MATHEMATICAL PROBLEMS

DNA computing is a rapidly growing field that utilizes DNA’s natural potential to spontaneously assemble and store information. DNA is also easy to manipulate which makes it adaptable to a wide range of applications. DNA computing uses this potential to solve computationally difficult mathematical problems. Creating a functional library of combinatorial strands of the required length is a prerequisite for a DNA computing model. This task is accomplished by using a computer algorithm that produces DNA sequences that will bind only to their complements. Validation of this algorithm was performed on test strand using SYBR Green I dye and a Real-time PCR machine to measure florescence. After validation was completed construction of the library was able to begin. We used a DNA polymerase-based method to combine ten unique, 16 base pair, single-stranded oligonucleotides, to from a library of 32 double-stranded molecules of length 80 base pairs.

Tumor Necrosis Factor Promotes Runx2 Degradation through Up-regulation of Smurf1 and Smurf2 in Osteoblasts

Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory bone loss through stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation. Compared with the well established role of TNF in osteoclastogenesis, mechanisms by which TNF inhibits osteoblast function have not been fully determined. Runx2 is an osteoblast-specific transcription factor whose steady-state protein levels are regulated by proteasomal degradation, mediated by the E3 ubiquitin ligases, Smurf1 and Smurf2. We hypothesized that TNF inhibits osteoblast function through Smurf-mediated Runx2 degradation. We treated C2C12 and 2T3 osteoblast precursor cell lines and primary osteoblasts with TNF and found that TNF, but not interleukin-1, significantly increased Smurf1 and Smurf2 expression. TNF increased the degradation of endogenous or transfected Runx2 protein, which was blocked by treating cells with a proteasomal inhibitor or by infecting cells with small interfering (si)RNA against Smurf1 or Smurf2. TNF inhibited the expression of bone morphogenetic protein and transforming growth factor-beta signaling reporter constructs, and the inhibition of each was blocked by Smurf1 siRNA and Smurf2 siRNA, respectively. Overexpression of Smurf1 and/or Smurf2 siRNAs prevented the inhibitory effect of TNF on Runx2 reporter. Consistent with these in vitro findings, bones from TNF transgenic mice or TNF-injected wild type mice had increased Smurf1 and decreased Runx2 protein levels. We propose that one of the mechanisms by which TNF inhibits bone formation in inflammatory bone disorders is by promoting Runx2 proteasomal degradation through up-regulation of Smurf1 and Smurf2 expression.

Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification

Optimal detection of a pathogen present in biological samples depends on the ability to extract DNA molecules rapidly and efficiently. In this paper, we report a novel method for efficient DNA extraction and subsequent real-time detection in a single microchip by combining laser irradiation and magnetic beads. By using a 808 nm laser and carboxyl-terminated magnetic beads, we demonstrate that a single pulse of 40 seconds lysed pathogens including E. coli and Gram-positive bacterial cells as well as the hepatitis B virus mixed with human serum. We further demonstrate that the real-time pathogen detection was performed with pre-mixed PCR reagents in a real-time PCR machine using the same microchip, after laser irradiation in a hand-held device equipped with a small laser diode. These results suggest that the new sample preparation method is well suited to be integrated into lab-on-a-chip application of the pathogen detection system.

262 ASSESSMENT OF HSP70-1 TRANSCRIPTION LEVELS IN IMMATURE OOCYTES FROM BOS TAURUS AND BOS INDICUS COWS RAISED IN A TROPICAL CLIMATE

Heat stress is one of the main causes of low conception rate in Bos taurus cows in a tropical climate. On the other hand, in this environment, oocytes from Bos indicus show greater developmental capacity after in vitro fertilization than those from Bos taurus, suggesting an adaptation to the hot climate. Heat shock proteins (HSP) are chaperones that promote protection against heat damage, and their transcription is associated to stress. The aim of this study was to evaluate the expression of HSP70-1 gene (Genbank NM174550), a member of HSP family, in oocytes from Bos taurus (Holstein) and Bos indicus (Gyr) cows raised in the tropical climate located at 21�35′′S latitude, 43�51′′W longitude, and 435 m altitude. Cumulus–oocyte complexes were recovered by oocyte pickup from mature non-lactating Holstein (n = 4) and Gyr (n = 4) donor cows during the hot season. Cumulus cells of viable oocytes were removed by vortexing in TALP-HEPES plus BSA, and pools (3 for each breed) with 12 immature oocytes were rapidly frozen in liquid nitrogen and subsequently thawed for RNA extraction. Total RNA extraction was performed using Rneasy� Micro kit (Qiagen, Valencia, CA, USA), and first strands were synthesized using SuperscriptTM III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using real-time PCR (ABI Prism� 7000; Applied Biosystems, Foster City, CA, USA); reactions consisted of a mixture of iTaqTM SYBR� Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) and cDNA equivalent to 1.2 oocytes and gene specific primers. Expression of the GAPDH gene was used as endogenous reference. Calculations of relative quantification were performed by the comparative Ct method, using the lowest value found in Bos indicus oocytes as calibrator; values (mean � SE) are shown as n-fold difference relative to the calibrator. Statistical comparison between breeds was performed by analysis of variance. Oocytes from Holstein cows showed a higher level (P &amp;lt; 0.05) of HSP70-1 expression (1.82 � 0.22) than oocytes recovered from Gyr cows (1.12 � 0.11). Previous study reported that oocytes from Gyr cows in a tropical climate showed a higher blastocyst rate after in vitro fertilization than Holstein oocytes (Camargo et al. 2006 Reprod. Fertil. Dev. 18, 243 abst). The lower level of HSP70-1 in Gyr oocytes suggests that they were less subject to stress than the Holstein ones, which may reflect their capacity to develop after fertilization. This effect may be, at least in part, due to the ability of Bos indicus cows to regulate body temperature in a hot environment, causing less stress on oocytes. Financial support was provided by FAPEMIG, MG, Brazil, and CNPq, DF, Brazil. Thanks to Agrogen�tica, Vi�osa, Brazil, for the real-time PCR machine.

280 EXPRESSION OF HSP70.1 GENE IN IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED IN CR2 MEDIUM SUPPLEMENTED WITH KNOCKOUT™SR

The exposure of embryos to serum during in vitro culture can affect morphology, metabolism, tolerance to cryopreservation, and expression of specific transcripts. On the other hand, serum-free medium seems to avoid some of those serum effects. KnockoutTMSR (GIBCO Laboratories, Grand Island, NY, USA) is a serum replacer optimized to support embryonic stem cells in culture and can also be used to replace serum during culture of in vitro-fertilized bovine embryos. The expression of genes associated with stress response, such as heat shock proteins (HSP), can be affected by in vitro culture conditions, being easily induced by a variety of stress agents, including culture medium components. This study aimed to determine whether KnockoutSR or serum in culture medium alters the relative abundance of HSP70.1 transcripts in in vitro-fertilized bovine embryos. Cumulus–oocyte complexes obtained from slaughterhouse ovaries were matured and feritlized in vitro. Presumptive zygotes were randomly cultured with their own cumulus cells in CR2aa medium supplemented with 10% fetal calf serum (GIBCO-BRL, Paisley, UK; FCS group), 10% KnockoutSR (GIBCO-BRL; KSR group), or 3 mg mL-1 of polyvinyl alcohol (PVA group). All steps were performed at 38.5�C, under 5% CO2 in air and 95% humidity. Blastocysts on Day 8 post-fertilization were rapidly frozen in liquid nitrogen and subsequently thawed for RNA extraction (3 replicates for each group). Total RNA extraction was performed using an Rneasy� Micro kit (Qiagen, Valencia, CA, USA), and the first strand was synthesized using SuperscriptTM III First Strand Synthesis kit (Invitrogen, Chicago, IL, USA). Relative quantification was performed in duplicate using real-time PCR (ABI Prism� 7000 Applied Biosystems, Foster City, CA, USA); reactions consisted of a mixture of iTaqTM SYBR� Green Supermix with ROX (Bio-Rad, Waltham, MA, USA) with cDNA equivalent to 0.8 embryos and gene-specific primers. Expression of the glyceraldehyde 3-phosphate dehydrogenase gene was used as endogenous reference. Calculations of relative quantification were performed by comparative Ct method, using the value found in the PVA group as calibrator. Expression levels for the FCS and KSR groups were 1.2 � 0.06- and 1.4 � 0.08-fold differences relative to the PVA group without differences (P &amp;gt; 0.05). These data show that bovine embryos cultured in medium supplemented with KSR have the same HSP70-1 expression pattern as those in medium with added FCS, suggesting that embryos in both groups are under the same stress conditions. This work was supported by FAPEMIG, MG, Brazil, and CNPq, DF, Brazil. Thanks to Agrogenetica, Vi�osa, Brazil, for the real-time PCR machine.

Chromatin Modification and the Endothelial-specific Activation of the E-selectin Gene

E-selectin plays a role in the binding and extravasation of leukocytes from the bloodstream. The E-selectin gene is rapidly and transiently expressed by endothelial cells activated by inflammatory stimuli. Despite the identification of factors critical for cytokine-induced activation of the E-selectin promoter, little is known about the mechanisms that restrict the gene expression to endothelial cells. We used in vivo approaches to characterize the E-selectin promoter in primary cultures of human umbilical vein endothelial cells and umbilical artery smooth muscle cells. In endothelial cells specifically, nucleosomes are remodeled after tumor necrosis factor (TNF) alpha induction. Chromatin immunoprecipitation analysis demonstrated the binding of the p65 (RelA) component of nuclear factor-kappa B (NF-kappa B) to the endogenous E-selectin promoter after TNFalpha stimulation along with IkappaB kinase alpha. Multiple coactivators, including p300, steroid receptor coactivator-1, and p300/cAMP-response element-binding protein (CREB)-binding protein (CBP)-associated factor localize differentially to the E-selectin promoter. Additionally, TNFalpha induced localized histone hyperacetylation, phosphorylation, and methylation in the E-selectin gene specifically in endothelial cells. Post-induction repression of E-selectin expression is associated with recruitment of multiple deacetylases. Collectively, these studies suggest a model for the selective induction of the E-selectin gene in which the core promoter chromatin architecture is specifically modified in endothelial cells.

Development of a CCD-based fluorimeter for real-time PCR machine

A monochromic charged coupled device (CCD) is employed to obtain time evolved fluorescence images that are reflected from PCR mix in a 20 μL capillary during thermal cycling in the real-time polymerase chain reaction (real-time PCR). This fluorescence detection system consists of a 470 nm light emitting diode (LED) as an excitation light source, a CCD camera, and a rotating disk with six optical filters to provide six fluorescence detection channels at 530, 560, 610, 640, 670 and 710 nm for multiplex real-time PCR DNA quantification. In this study, the PCR mix that contains HBV SC 11 DNA template with SYBR Green I fluorescence labeling dye is tested to verify the prototype performance. An adaptive image processing scheme based on a lower–upper–middle (LUM) filter algorithm is developed to eliminate noise while keeping the necessary information from recorded fluorescence images. The measured results obtained by the proposed image processing scheme illustrate that the real-time PCR prototype with CCD-based fluorimeter can achieve similar DNA quantification reproducibility as the commercial machine even when the initial DNA copies, X, in the tested PCR mix is down to 10 3 copies/mL (0.01 fM).

5 ASSESSMENT OF HSP70.1 TRANSCRIPTION LEVELS IN BOVINE EMBRYOS AFTER CUMULUS REMOVAL BY DIFFERENT TECHNIQUES

Previous results indicated that there was a difference in transcriptional activity depending on the method used to remove the cumulus cells after IVF (Zeringue et al. 2005 LabChip 5, 86-90). However, specific gene expression was not examined and therefore was the goal of the present study. The objective of this study was to compare the transcription levels of the HSP70.1 gene in bovine in vitro production (IVP) embryos that underwent cumulus removal by vortexing, hand stripping, or microfluidic treatment. Quantitative real-time PCR was used to estimate transcription levels of the HSP70.1 gene in bovine IVP zygotes and two-cell embryos. Transcription levels were compared between embryos that underwent three methods of cumulus removal. Presumptive zygotes were harvested at 2 or 24 h after cumulus removal by vortexing, by hand stripping, or by microfluidic means in order to compare the relative embryonic stress of these three treatments. Bovine in vitro embryos were produced by standard means with the only variable being the cumulus removal technique 24 h after fertilization. At 2 and 24 h post-cumulus removal, randomly selected presumptive zygotes were taken out of culture, preserved in RNAlater (Ambion, Inc., Austin, TX, USA) and stored at -20�C. RNA was extracted from single embryos via Qiagen's RNeasy Micro kit (Valencia, CA, USA). RNA was amplified and PCR products were detected with SYBR Green 1 (Applied Biosystems, Foster City, CA, USA) using an Opticon Monitor 3 real-time PCR machine (Bio-Rad Laboratories, Inc., Waltham, MA, USA). The threshold cycle (CT) numbers were determined for the amplified cDNA of the bovine HSP70.1 mRNA and for the housekeeping gene, acidic ribosomal phosphoprotein (PO), used as a reference. Then the amount of HSP70.1 was divided by the amount of PO to obtain a normalized HSP70.1 value expressed as the ratio of HSP70.1/PO. Ratios were analyzed by the GENMOD procedure in SAS (SAS Institute, Inc., Cary, NC, USA) accounting for replicates and treatments. Transcription levels of the HSP70.1 gene did not differ significantly between the microfluidically treated and vortexed groups of zygotes at 2 h post-cumulus removal (P = 0.1032) or between the hand-stripped and vortexed groups (P = 0.7567). In contrast, at 24 h post-cumulus removal, the embryos in the microfluidically treated group showed significantly higher levels of HSP70.1 transcription than the vortexed group (P = 0.0115). The transcription levels did not differ significantly between the hand-stripped and vortexed groups (P = 0.7875). This work strongly suggests that there is de novo RNA transcription in the early embryonic stages of the bovine. In addition to previously described improved developmental kinetics, the use of microfluidics in IVP leads to statistically significant differences in RNA transcription levels of HSP70.1.

Evaluation of real-time quantitative PCR machines for the monitoring of fusion gene transcripts using the Europe against cancer protocol

Evaluation of real-time quantitative PCR machines for the monitoring of fusion gene transcripts using the Europe against cancer protocol

OPTIMISATION OF RELATIVE QUANTITATIVE RT-PCR FOR EXPRESSION ANALYSIS IN AZALEA FLOWER COLOUR SPORTS

The fastest way to create new azalea (Rhododendron simsii hybrids) cultivars is by making use of flower colour sports, which appear spontaneously on azalea plants. Unfortunately, there is still very little known on how bud sport induction occurs. Therefore, genes coding for two key enzymes of the azalea flavonoid biosynthesis pathway, chalcon synthase (chs) and dihydroflavonol 4-reductase (dfr) that were reported before to be apt for modification by the action of bud sporting, were isolated and characterized. The expression of these two flower colour genes in the petals of azalea flowers will be compared between all ‘Hellmut Vogel’ flower colour sports. To measure the expression levels of both genes, relative quantitative RT-PCR analysis will be worked out on a real-time PCR machine. The expression of housekeeping genes, which is expected to be the same for all sports, will be used to calculate the relative expression level of the two genes of interest. The optimisation of this technique will be discussed.

A novel real-time PCR machine with a miniature spectrometer for fluorescence sensing in a micro liter volume glass capillary

This study sets up a prototype of real-time polymerase chain reaction (RT-PCR) machine that employs a miniature spectrometer for detecting the emission of fluorescence intensity from RT-PCR mix in a micro liter volume glass capillary. The RT-PCR machine is one of the major instruments for SARS virus test during the outbreak in Asia in early 2003. Comparing with traditional RT-PCR machine with discrete channels fluorescence wavelength detection, the prototype can provide continuous wavelength detection and can be employed for multiplex DNA quantification. However, only one HBV SC 11 DNA template with the SYBR Green I labeling dye were used in this study to compare DNA quantification accuracy and reproducibility of the present system and the commercial system. The two machines have different optical engine arrangement and so two separate analytical models were proposed to predict the fluorescence intensity from the RT-PCR mix during thermal cycling for the machines. Predicted results agree well with the measured data for both machines. From the predicted results, different approaches should be adopted to determine the initial DNA concentration for quantification from time recorded history of the fluorescence intensity. Measured results illustrate that the RT-PCR prototype has the same accuracy for DNA quantification and reproducibility within five intra-assay samples as compared with the commercial machine.

Real-time polymerase chain reaction.

Real-time PCR is the state-of-the-art technique to quantify nucleic acids for mutation detection, genotyping and chimerism analysis. Since its development in the 1990s, many different assay formats have been developed and the number of real-time PCR machines of different design is continuously increasing. This review provides a survey of the instruments and assay formats available and discusses the pros and cons of each. The principles of quantitative real-time PCR and melting curve analysis are explained. The quantification algorithms with internal and external standardization are derived mathematically, and potential pitfalls for the data analysis are discussed. Finally, examples of applications of this extremely versatile technique are given that demonstrate the enormous impact of real-time PCR on life sciences and molecular medicine.

Comparison between TaqMan and LightCycler technologies for quantification of minimal residual disease by using immunoglobulin and T-cell receptor genes consensus probes

Quantification of residual leukemic cells at early time points during therapy can reliably predict the outcome in children with acute lymphoblastic leukemia (ALL). Recently, semiquantitative minimal residual disease (MRD) detection assays such as dot-blot hybridization have been replaced by real-time quantitative PCR. We tested the flexibility of the two most used real-time PCR machines: the SDS 7700 or 'TaqMan' (TM) (Applied Biosystems) and the LightCycler (LC) (Roche) instruments. Clonal T-cell receptor and immunoglobulin gene rearrangements were used for MRD detection with germline hydrolyzation probes and clone-specific primers. Sensitivity tests for 65 clonal gene rearrangements and MRD quantification in 90 bone marrow samples during therapy of 49 children with ALL at diagnosis or relapse were performed with both machines. Both real-time PCR systems provided specific results for MRD quantification in all follow-up samples. In conclusion, we were able to demonstrate that TM and LC real-time PCR technologies produce similar MRD quantification results and that the quantification assays can be easily transferred from one detection system to the other. Using the same detection format, both techniques can be applied in combination in multicenter MRD studies.

Improving the accuracy of genetic diagnosis from single cells through analysis of real-time PCR signal kinetics

Improving the accuracy of genetic diagnosis from single cells through analysis of real-time PCR signal kinetics

Evaluation of the portable Cepheid SmartCycler real-time PCR machine for the rapid diagnosis of foot-and-mouth disease

The ability of the portable Cepheid SmartCycler real-time PCR machine to detect foot-and-mouth disease (FMD) virus sensitively and accurately was evaluated by comparing the results of the analyses of nasal...