SYBR Green System Research Articles

Development of real-time PCR methods for the quantification of Methanoculleus, Methanosarcina and Methanobacterium in anaerobic digestion

Anaerobic digestion is a growing technology to manage organic waste and produce bioenergy. To promote this technology, it is essential to know, at the molecular level, the dynamics of microbial communities, specifically the methanogenic community. In the present study, three primer pairs were selected from seven primer pairs which were designed and tested with different concentrations and conditions to detect Methanosarcina, Methanoculleus and Methanobacterium by real-time PCR based on the SYBR Green System. The functionality of the developed methods was demonstrated by the high linear relationship of the standard curves, and the specificity of each primer was empirically verified by testing DNA isolated from methane-producing and non-producing strains. These assays also exhibited good repeatability and reproducibility, which indicates the robustness of the methods. The described primers were successfully used to investigate the methanogenic communities of 10 samples from an anaerobic co-digestion. The genus Methanosarcina was the dominant methanogenic group.

Evaluation of quality and gene expression of goat embryos produced in vivo and in vitro after cryopreservation

In the present study, we aimed to identify morphological and molecular changes of in vivo and in vitro-produced goat embryos submitted to cryopreservation. In vivo embryos were recovered by transcervical technique from superovulated goats, whereas in vitro produced embryos were produced from ovaries collected at a slaughterhouse. Embryos were frozen by two-steps slow freezing method, which is defined as freezing to −32 °C followed by transfer to liquid nitrogen. Morphological evaluation of embryos was carried out by assessing blastocoel re-expansion rate and the total number of blastomeres. The expression profile of candidate genes related to thermal and oxidative stress, apoptosis, epigenetic, and implantation control was measured using RT-qPCR based SYBR Green system. In silico analyses were performed to identify conserved genes in goat species and protein-protein interaction networks were created. In vivo-produced embryos showed greater blastocoel re-expansion and more blastomere cells (P < 0.05). The expression level of CTP2 and HSP90 genes from in vitro cryopreserved embryos was higher than their in vivo counterparts. Unlikely, no significant difference was observed in the transcription level of SOD gene between groups. The high similarity of CPT2 and HSP90 proteins to their orthologs among mammals indicates that they share conserved functions. In summary, cryopreservation negatively affects the morphology and viability of goat embryos produced in vitro and changes the CPT2 and HSP90 gene expression likely in response to the in vitro production process.

Profile of Enterobacteria Resistant to Beta-Lactams.

A serious emerging problem worldwide is increased antimicrobial resistance. Acquisition of coding genes for evasion methods of antimicrobial drug mechanisms characterizes acquired resistance. This phenomenon has been observed in Enterobacteriaceae family. Treatment for bacterial infections is performed with antibiotics, of which the most used are beta-lactams. The aim of this study was to correlate antimicrobial resistance profiles in Enterobacteriaceae by phenotypic methods and molecular identification of 14 beta-lactamase coding genes. In this study, 70 exclusive isolates from Brazil were used, half of which were collected in veterinary clinics or hospitals Phenotypic methodologies were used and real-time PCR was the molecular methodology used, through the Sybr Green system. Regargding the results found in the tests it was observed that 74.28% were resistant to ampicillin, 62.85% were resistant to amoxicillin associated with clavalunate. The mechanism of resistance that presented the highest expression was ESBL (17.14%). The genes studied that were detected in a greater number of species were blaGIM and blaSIM (66.66% of the samples) and the one that was amplified in a smaller number of samples was blaVIM (16.66%). Therefore, high and worrying levels of antimicrobial resistance have been found in enterobacteria, and a way to minimize the accelerated emergence of their resistance includes developing or improving techniques that generate diagnoses with high efficiency and speed.

Microbial analysis of root canal and periradicular lesion associated to teeth with endodontic failure

The quantification of ten microorganisms at the root ends and in the surrounding periradicular lesions was performed. Thirty 3 mm samples root ends and 30 samples of the surrounding chronic periapical infection were collected during apical microsurgery. Samples were triturated, and the bacterial DNA was obtained. The bacterial quantification was performed by using the SYBR Green system. At least one microorganism was detected in all patients. In both the root end and periapical samples, Fusobacterium nucleatum (71.6%), Dialister pneumosintes (58.3%) and Tannerella forsythia (48.3%) were the most prevalent species. Dialister pneumosintes showed statistically significant values in the root end, and F. nucleatum was also significant in the apical periodontitis samples. A statistically significant association between T. forsythia and Porphyromonas gingivalis in the root ends was observed. Bacterial associations from 2 to 7 species were observed in most samples. Extra-radicular and/or intra-radicular infections were present in all teeth with failed endodontic treatment, and showed polymicrobial infection in most cases, with a predominance of F. nucleatum, D. pneumosintes and T. forsythia. When present, Enterococcus faecalis was never found to be the most prevalent species. The presence of a microbial diversity in post-treatment apical periodontitis confirms the polymicrobial and synergistic characteristic of this process. Our results show that the bacterial array associated with the 3 mm root ends and periradicular lesions in post-treatment apical periodontitis are complex and with a high inter-individual variability. These results might be useful to delineate treatment strategies for microbial elimination in apical periodontitis. Further studies are necessary to elucidate the role of these microorganisms in endodontic treatment failures.

A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR.

Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology.

Detection of five potentially periodontal pathogenic bacteria in peri-implant disease: A comparison of PCR and real-time PCR

The aim of this study was to compare the microbial analysis methods of polymerase chain reaction (PCR) and real-time PCR (RT-PCR) in terms of detection of five selected potentially periodontal pathogenic bacteria in peri-implant disease. Therefore 45 samples of healthy, mucositis and peri-implantitis (n = 15 each) were assessed according to presence of the following bacteria using PCR (DNA-strip technology) and RT-PCR (fluorescent dye SYBR green-system): Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tanerella forsythia (Tf), and Fusobacterium nucleatum (Fn). There were no significant correlations between the bacterial and disease patterns, so the benefit of using microbiological tests for the diagnosis of peri-implant diseases is questionable. Correlations between the methods were highest for Tf (Kendall's Tau: 0.65, Spearman: 0.78), Fn (0.49, 0.61) and Td (0.49, 0.59). For Aa (0.38, 0.42) and Pg (0.04, 0.04), lower correlation values were detected. Accordingly, conventional semi-quantitative PCR seems to be sufficient for analyzing potentially periodontal pathogenic bacterial species.

Association between periodontal condition and subgingival microbiota in women during pregnancy: a longitudinal study

Objectivo In this study, the gingival conditions and the quantitative detection for Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia in pregnant women were determined.Material and Methods Quantitative determinations of periodontal bacteria by using a SyBr green system in women during pregnancy were performed. Women at the 2nd and 3rd trimesters of pregnancy and non-pregnant women were included in this study. A. actinomycetemcomitans was observed in high numbers in women at the 2nd and 3rd trimesters of pregnancy with a significant difference (p<0.05). F. nucleatum and P. intermedia were also observed in high levels.Results and Conclusion Our results show that pregnant women are more susceptible to gingivitis, and the presence of A. actinomycetemcomitans in subgingival biofilm might be taken into account for the treatment of periodontal disease.

Comparison between Conventional and Real-Time PCR Assays for Diagnosis of Visceral Leishmaniasis

The diagnosis of visceral leishmaniasis (VL) is a challenging issue and several studies worldwide have evaluated the different tools to reach a diagnostic solution. The polymerase chain reaction (PCR) has proven to be effective in detecting the genome of Leishmania species in different biological samples. In this study, we compared the conventional PCR and real-time PCR using the Sybr Green system and their application in molecular diagnosis of visceral leishmaniasis in peripheral blood as a biological sample. The genus-specific conserved region of kinetoplast DNA (kDNA) was the target of amplification. We studied 30 samples from patients with suspect of visceral leishmaniasis who were treated by the Medical Clinic of Santa Casa de Belo Horizonte Hospital, Brazil. Among the samples studied, 19 had a confirmed diagnosis for VL by serology and/or by clinical findings. Among these 19 samples, 63% (n = 12) presented positive results for serology and 79% (n = 15) positive results in both PCR methodologies. This fact suggests that the PCR technique can assist in the diagnosis of visceral leishmaniasis in patients who do not have detectable antibodies by serology but can present the genome of the parasite circulating in whole blood. Also, it was possible to observe that there was conformity between the results of the techniques of cPCR and qPCR using the Sybr Green system in 100% of samples analyzed. These data suggest that both PCR techniques were equally effective for detection of the genome of the parasite in the patient's blood.

178 EXPRESSION OF mRNA ENCODING LUTEINIZING HORMONE RECEPTOR AND MEVALONATE KINASE AROUND FOLLICLE DEVIATION IN NELORE HEIFERS (BOS INDICUS)

Luteinizing hormone (LH) plays a key role in controlling physiological processes in the ovary, and the expression of LHR by bovine granulosa cells is crucial to the follicular transition from FSH to LH dependency. There are controversies about the time at which follicles acquire LHR in granulosa cells. In Nelore breed (Bos indicus), the morphological divergence occurs, on average, 2.5 days after ovulation when the diameter of the dominant follicle is ~6.0 mm. In our previous work with semiquantitative PCR, the mRNA expression of LHR isoforms was detected more clearly after deviation (Day 3). The LHR mRNA binding protein, mevalonate kinase (MVK), is responsible for the down-regulation of LHR mRNA, thereby controlling the steady-state of LHR mRNA expression. In rats, there is an inverse correlation between the mRNA expression of LHR and MVK in luteal cells; however, there is no evidence about MVK expression in the bovine antral follicle. To gain insight about the involvement of the LHR/MVK system in the control of follicle deviation, we assessed the mRNA expression of LHR and MVK in granulosa cells from dominant and subordinate follicles close to deviation in Nelore heifers. Animals (n = 10) were hormonally synchronized, and ovulation was detected by ultrasound monitoring every 12 h. Heifers were slaughtered 2 (before deviation; n = 3), 2.5 (around deviation; n = 4), and 3 (post-deviation; n = 3) days after ovulation. Granulosa cells were harvested from the 2 largest follicles and submitted to total RNA extraction and reverse transcribed with oligo-dT. The mRNA abundance of LHR and MVK was measured by real-time RT-PCR using the Sybr Green system with bovine-specific primers and normalized by the expression of endogenous gene, cyclophilin A (PPIA), using the ΔΔct method corrected by Pffafl’s equation. Dominant and subordinate follicles were considered those expressing the greatest and second greatest abundance of aromatase mRNA (CYP19) in granulosa cells within each heifer. Effects of the day and follicle status on the mRNA abundance of LHR and MVK were tested by ANOVA and the mean values compared by paired t-test or Tukey test (P &lt; 0.05 indicated significant difference). The LHR mRNA was detected at the predicted time of follicle deviation in Nelore heifers (Day 2.5) and was higher in dominant follicle on Day 3 (32.8 ± 12.6) compared with Day 2.5 (3.2 ± 0.9). The second largest follicle (subordinate follicles) had lower mRNA abundance of LHR when compared with future dominant follicles (largest follicles) on days 2.5 (0.8 ± 0.4 v. 3.2 ± 0.9) and 3 (1.9 ± 0.8 v. 32.8 ± 12.6). In contrast to the mRNA expression of LHR, MVK mRNA was more expressed in the subordinate follicles than in the largest follicles at Days 2.5 (3.1 ± 0.9 v. 0.9 ± 0.3) and 3 (2.6 ± 0.6 v. 0.9 ± 0.1) after ovulation, suggesting that it may be necessary to decrease the MVK expression in future dominant follicles to increase their LHR expression and follow up to ovulation. Supported by FAPESP.

A versatile real-time PCR method to quantify bovine contamination in buffalo products

Fraudulent species substitution in food products is a reality in many markets around the world. The consequences are serious and impact governments, consumers and producers. Molecular methods, including PCR-based techniques, are available to prevent species substitutions and to authenticate food content. Here, we describe a method for calculating the bovine and buffalo content in milk- and meat-derived food products. The method applies real-time PCR using primers designed to specifically amplify bovine or buffalo DNA. The amplification efficiencies of both primer sets were assessed using the TaqMan and SYBR Green systems. Both sets of primers showed satisfactory results. A quantification procedure is proposed and involves amplifying a sample with both primer sets and then normalizing the total DNA using the total non-normalized bovine and buffalo DNA. To correct for the potential deviations between the real and measured DNA quantity caused by biological differences between species, we propose the use of calibration curves generated from each analyzed matrix. These curves include a set of controlled admixtures of bovine and buffalo material. Using this method for dairy samples containing known bovine and buffalo content, the use of the calibration curve always approximated the measured to the expected quantities. This technique was then tested on commercial samples. Our method was efficacious and reliable and can be applied to the routine analysis of these products. Although we only tested the methodology on dairy products, it can also be applied to other food matrices, such as meat-derived products. A patent document for the technique has been submitted to the Brazilian Patent Office (INPI – Instituto Nacional da Propriedade Industrial –www.inpi.gov.br) under the number 014110002844.

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.

Functional Mutation of SMAC/DIABLO, Encoding a Mitochondrial Proapoptotic Protein, Causes Human Progressive Hearing Loss DFNA64

SMAC/DIABLO is a mitochondrial proapoptotic protein that is released from mitochondria during apoptosis and counters the inhibitory activities of inhibitor of apoptosis proteins, IAPs. By linkage analysis and candidate screening, we identified a heterozygous SMAC/DIABLO mutation, c.377C>T (p.Ser126Leu, refers to p.Ser71Leu in the mature protein) in a six-generation Chinese kindred characterized by dominant progressive nonsyndromic hearing loss, designated as DFNA64. SMAC/DIABLO is highly expressed in human embryonic ears and is enriched in the developing mouse inner-ear hair cells, suggesting it has a role in the development and homeostasis of hair cells. We used a functional study to demonstrate that the SMAC/DIABLO(S71L) mutant, while retaining the proapoptotic function, triggers significant degradation of both wild-type and mutant SMAC/DIABLO and renders host mitochondria susceptible to calcium-induced loss of the membrane potential. Our work identifies DFNA64 as the human genetic disorder associated with SMAC/DIABLO malfunction and suggests that mutant SMAC/DIABLO(S71L) might cause mitochondrial dysfunction.

Detection of Anaplasma phagocytophilum in Brazilian dogs by real-time polymerase chain reaction

Anaplasma phagocytophilum was detected in dogs from Brazil in the municipalities of Seropédica and Itaguaí, Rio de Janeiro state, by real-time polymerase chain reaction (PCR) using SYBR Green to detect the amplification. Of 253 samples, 18 (7.11%) were positive, with a threshold cycle (Ct) ranging from 31 to 35 cycles. The PCR product from a positive sample was cloned and sequenced. The sequence obtained demonstrated 100% identity with other A. phagocytophilum sequences published in the GenBank database. The analytical sensitivity of RT-PCR using SYBR Green system was able to detect 3 plasmid copies when defined numbers of plasmid copies containing 122 base pairs from the msp2 gene were used. The assay was considered specific when DNA from bacteria (Anaplasma platys, Anaplasma marginale, Ehrlichia canis, Neorickettsia risticii, Rickettsia rickettsii) closely related to A. phagocytophilum was placed in the reaction. These results demonstrate that the canine granulocytic anaplasmosis agent is present in regions in which dogs could be a source of infection for tick vectors. The current study reports the detection of A. phagocytophilum, a zoonotic agent responsible for Human granulocytic anaplasmosis, in Brazilian dogs.

ASPM gene expression in medulloblastoma

Medulloblastomas are the most common malignant tumors of the central nervous system in childhood. The incidence is about 19-20% between children younger than 16 years old with peak incidence between 4 and 7 years. Despite its sensibility to no specific therapeutic means like chemotherapy and radiotherapy, the treatment is very aggressive and frequently results in regression, growth deficit, and endocrine dysfunction. From this point of view, new treatment approaches are needed such as molecular targeted therapies. Studies in glioblastoma demonstrated that ASPM gene was overexpressed when compared to normal brain and ASPM inhibition by siRNA-mediated inhibits tumor cell proliferation and neural stem cell proliferation, supporting ASPM gene as a potential molecular target in glioblastoma. The aim of this work was to evaluate ASPM expression in medulloblastoma fragment samples, and to compare the results with the patient clinical features. Analysis of gene expression was performed by quantitative PCR real time using SYBR Green system in tumor samples from 37 children. The t test was used to analyze the gene expression, and Mann-Whitney test was performed to analyze the relationship between gene expressions and clinical characteristics. Kaplan-Meier test evaluated curve survival. All samples overexpressed ASPM gene more than 40-fold. However, we did not find any association between the overexpressed samples and the clinical parameters. ASPM overexpression may modify the ability of stem cells to differentiate during the development of the central nervous system, contributing to the development of medulloblastoma, a tumor of embryonic origin from cerebellar progenitor cells.

Fetal Sex Determination of Macaque Monkeys by a Nested PCR Using Maternal Plasma

Non-invasive fetal sex determination is required for biomedical studies, in which some sexual difference would be expected in fetal events, in order to make a choice of male or female fetus. To detect male fetal DNA of the sex-determining region Y gene (SRY) in maternal macaque plasma, nested real-time PCR using the SYBR Green system was developed. In all cases of pregnant macaques with male fetuses, a nested PCR product of SRY was amplified from the mother's plasma, while no amplicon was detected in any case of pregnancy with a female fetus. Interestingly, fetal SRY DNA appeared to be cleared rapidly from the maternal blood after parturition. The current method is sensitive and can be performed with a regular PCR machine.

SGK1 Phosphorylation of IκB Kinase α and p300 Up-regulates NF-κB Activity and Increases N-Methyl-D-aspartate Receptor NR2A and NR2B Expression

Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a downstream target of phosphatidylinositol 3-kinase signaling, and it regulates various cellular and physiological functions, but the SGK1 substrate proteins and genes regulated by SGK1 are less known. Here we have identified IkappaB kinase alpha (IKKalpha) as a novel substrate of SGK1 by using biochemical and bioinformatic approaches. SGK1 directly phosphorylates IKKalpha at Thr-23 and indirectly activates IKKalpha at Ser-180. Furthermore, SGK1 enhanced nuclear factor kappaB (NF-kappaB) activity and up-regulated N-methyl-d-aspartate receptor NR2A and NR2B expression through activation of IKKalpha at Thr-23 and Ser-180, and these two residues play an equally important role in mediating these effects of SGK1. Although SGK1 does not phosphorylate IKKbeta, IKKbeta activity is still required for IKK complex activation and for SGK1 phosphorylation and activation of NF-kappaB. In addition, SGK1 increased the acetylation of NF-kappaB through phosphorylation of p300 at Ser-1834, and this also leads to NF-kappaB activation and NR2A and NR2B expression. Moreover, an endogenous stimulus of SGK1, insulin, increased IKKalpha and NF-kappaB phosphorylation as well as NF-kappaB acetylation and NF-kappaB activity, but SGK1 small interfering RNA transfection blocked these effects of insulin. In examination of the functional significance of the SGK1-IKKalpha-NF-kappaB signaling pathway, we found that transfection of the IKKalpha double mutant (IKKalphaT23A/S180A) to rat hippocampus antagonized SGK-1-mediated spatial memory facilitation. Our results together demonstrated novel substrate proteins of SGK1 and novel SGK1 signaling pathways. Activation of these signaling pathways enhances NR2A and NR2B expression that is implicated in neuronal plasticity.

A real-time PCR assay for the monitoring of influenza a virus in wild birds

A screening system including a new real-time PCR assay for the monitoring of influenza A virus in wild birds was developed. The real-time PCR assay uses SYBR green chemistry and the primers are targeting the matrix gene of influenza A virus. The performance of the assay was compared with two other assays, one assay also using SYBR green chemistry and one assay using TaqMan chemistry, i.e. a specific probe. A total of 45 fecal bird samples were analysed for influenza A virus in three different PCR reactions. Overall, 26 samples were positive in at least one of the three real-time PCR assays. Of the 26 samples, 18 were positive by all three reactions. Eight samples were found positive exclusively by the two SYBR green reactions, six of which were detected by both SYBR green reactions. Of the 26 positive samples, 15 samples were verified as positive either by virus isolation or influenza A M2-gene PCR. The results showed that the two SYBR green systems had a higher performance regarding the detection of influenza A as compared to the PCR reaction using a specific probe.

SYBR green and TaqMan real‐time PCR assays are equivalent for the diagnosis of dengue virus type 3 infections

Dengue is the most important arthropod-borne viral disease in the world. A rapid diagnostic test for dengue is warranted, and real-time polymerase chain reaction may improve diagnosis. TaqMan and Sybr Green systems were evaluated for the diagnosis of dengue virus type 3 (DENV-3) infections. Out of 77 patients with clinical suspicion of dengue infection, specific IgM antibodies were detected in 40 patients. DENV-3 was detected and quantitated in 17 IgM-positive samples by both systems. These assays were shown to be rapid, and specific for detection of DENV-3, and that for diagnostic purposes, there is no difference between these two assays.

Specific Structural Features of Heparan Sulfate Proteoglycans Potentiate Neuregulin-1 Signaling

Neuregulins are a family of growth and differentiation factors that act through activation of cell-surface erbB receptor tyrosine kinases and have essential functions both during development and on the growth of cancer cells. One alternatively spliced neuregulin-1 form has a distinct heparin-binding immunoglobulin-like domain that enables it to adhere to heparan sulfate proteoglycans at key locations during development and substantially potentiates its activity. We examined the structural specificity needed for neuregulin-1-heparin interactions using a gel mobility shift assay together with an assay that measures the ability of specific oligosaccharides to block erbB receptor phosphorylation in L6 muscle cells. Whereas the N-sulfate group of heparin was most important, the 2-O-sulfate and 6-O-sulfate groups also contributed to neuregulin-1 binding in these two assays. Optimal binding to neuregulin-1 required eight or more heparin disaccharides; however, as few as two disaccharides were still able to bind neuregulin-1 to a lesser extent. The physiological importance of this specificity was shown both by chemical and siRNA treatment of cultured muscle cells. Pretreatment of muscle cells with chlorate that blocks all sulfation or with an siRNA that selectively blocks N-sulfation significantly reduced erbB receptor activation by neuregulin-1 but had no effect on the activity of neuregulin-1 that lacks the heparin-binding domain. These results suggest that the regulation of glycosaminoglycan sulfation is an important biological mechanism that can modulate both the localization and potentiation of neuregulin-1 signaling.

T cell receptor BV repertoires using real time PCR: a comparison of SYBR green and a dual-labelled HuTrec™ fluorescent probe

Real time PCR is a useful tool in immunological research but little has been published on the use of this technique in the measurement of T cell receptor (TCR) BV repertoires. We have compared the performance of SYBR green with that of a dual-labeled HuTrec™ (Human T cell receptor) fluorescent probe system. Serial dilutions of peripherals blood mononuclear cells were tested to compare the consistency of the two systems across multiple T cell receptor signal levels. Samples were diluted with non-TCR cDNA to simulate a low-level TCR signal within a tissue sample. The fluorogenic probe gave highly consistent results with a correlation coefficient of greater than 0.9 across a samples. SYBR green showed accurate results only when tested with high signal samples. Low-signal cDNA gave very poor results with SYBR green compared to the HuTrec™ fluorogenic probe with correlation coefficients as low as 0.65. Poorest performance occurred in the context of the simulated tissue sample with a high level of non-TCR DNA. Under these conditions, large amounts of nonspecific PCR product were generated which were detected by the SYBR green system and therefore distorted the results. SYBR green performed poorly when used with samples contaminated with significant quantities of non-target cDNA and samples with low target signal. It is therefore not an appropriate method for the measurement of TCR repertoires in small tissue samples. A dual-labeled HuTrec™ fluorescent probe produced a consistent TCR repertoire across a broad range of TCR signal levels and proved robust in the presence of contaminating non-TCR cDNA. We recommend the use of such a fluorescent probe in real time PCR for the assessment of TCR repertoires in small tissue samples. Where samples are assayed using SYBR green, agarose gel confirmation of PCR product specificity should be provided.