Development and validation of real-time fluorescence quantitative PCR method for murine virus / 中国生物制品学杂志

@#Objective To develop and validate the real-time fluorescent quantitative PCR(Q-PCR)method for the detection of 8 murine viruses. Methods The specificity,sensitivity and precision of the Q-PCR method were verified by four laboratories,and the virus simulated contamination test and blind sample detection were carried out simultaneously,of which the detection results were compared. The Q-PCR method was used to detect 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other products of murine origin. Results The Q-PCR method used for detecting 8 kinds of murine viruses had no cross reaction with the same family and genus or other murine viruses. Except the sensitivity of laboratory 2 to ectromelia virus(EctV/Mouse Pox,MPV)was 2 × 10~2copies/μL,the sensitivity of laboratory 2 to other 7 viruses and 3 other laboratories to 8 murine viruses was 2 × 10~1copies/μL. Except the inter-assay CV of the copy number of mouse adenovirus(MAdV)detected by laboratory 3 was 37. 58%,the intra-assay and inter-assay CVs of the Ct and copy number of other 7 viruses detected by laboratory 3 and those of 8 viruses detected by other 3 laboratories were all less than 25%.The sensitivity of virus simulated contamination test met the parameter requirements. The coincidence rate of blind sample detection results by 4 laboratories was 100%. All the 26 batches of monoclonal antibody cell lines for SARS-CoV-2 vaccine production and 15 batches of other murine derived products were negative for 8 murine viruses. Conclusion Q-PCR method for murine virus has good specificity,sensitivity and precision,and can be used for the detection of murine derived biological products.

Formulation Process Analysis of a Virus-based Biopesticide to Control the Tomato leafminer Tuta absoluta

Abstract: Virus-based biopesticides are effective biocontrol agents of crop insect pests. Development of suitable formulations and production processes are necessary to obtain high-quality products easily adopted by farmers. A detailed unit operation study was carried out for the production process of a Phthorimaea operculella granulovirus-based biopesticide to control the tomato leafminer, Tuta absoluta, one of the most important pests affecting this crop. Physicochemical, microbiological, and insecticidal parameters were implemented in the process and applied to the finished product, and a scaling strategy was developed. A Quantitative Polymerase Chain Reaction (Q-PCR) technique was implemented to quantify viral concentrations in the active ingredient (5.34 ± 1.44 x109 Occlusion Bodies mL-1) and in the finished product (>1.6x109 OB mL-1), without contaminant interferences. The Q-PCR methodology was also useful to select the appropriate solid mixing time following Lacey´s mixing index (8 min). Factors and similarity principles influencing the liquid mixing process were identified in the scaling evaluation. Furthermore, the drying kinetics analysis enabled identifying a drying temperature of 35 °C, with an efficacy under controlled conditions higher than 97%. Contaminant concentration was lower than 1%, indicating controlled and aseptic formulation process conditions. A simple statistical method was used to estimate the reproducibility and repeatability of the parameters assessed in the finished product. These results enable to establish and extrapolate important parameters in the standardization, scale-up, and quality control for the granulovirus-based biopesticide.

Analysis of transcriptome sequencing and related genes of flavonoid biosynthesis from Anoectochilus roxburghii / 中国药科大学学报

@#Transcriptome sequencing was performed for the first time on Anoectochilus roxburghii(AR)in different harvesting periods using RNA-seq high-throughput sequencing technique, and the results were verified and analyzed by Q-PCR and HPLC. A total of 51, 370 genes were obtained by transcriptome sequencing and annotated to the database of Nr, GO, Swiss-Prot, KEGG and KOG. The species that were sequenced according to the homology sequence were the same as AR monocotyledon plants. Through comparison of AR transcriptome in different periods, it was found that the differences were mainly in flavonoid biosynthesis-related genes. The expression levels of flavonoid biosynthesis-related genes(trans-cinnamate 4-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, flavonol synthase, shikimate O-hydroxycinnamoyltransferase and flavonoid 3′, 5′-hydroxylase)were verified by Q-PCR, and the results were consistent with those of transcriptome sequencing. The contents of 6 flavonoids(rutin, isoquercitrin, narcissin, quercetin, kaempferol and isorhamnrtin)were determined by HPLC. The results showed that the expression of flavonoid synthetic gene in AR increased with the growth time, and the variation trend of flavonoid compound content and gene expression were basically consistent. Combined with transcriptome data, the biosynthetic pathway of flavonoid content in AR was plotted. This study provides important genetic resources for the key genes of flavonoid synthesis in AR and the biosynthesis of flavonoids, as well as the basis for the development of its medicinal value.

Expression of thymidylate synthase in breast cancer and its correlation with fluorouracil sensibility and patients' prognosis / 中华内分泌外科杂志

Objective To investigate the expression of thymidylate synthase (TS) in breast cancer and its relationship with fluorouracil sensibility and patients' prognosis.Methods TS expression in 80 cases of breast cancer was measured by RT-QPCR.The chemotherapy (CAF) was given.Relationship between TS and fluorouracil sensibility was studied,as well as the relation between TS expression and prognosis of breast cancer.Result Positive expression of TS gene was detected by Q-PCR 1n 27.5％ of the breast cancer specimens.The expression of TS had no relation with age,lymphatic metastasis,histological grade or clinical stage,while it was obviously correlated with HER2 (P＜0.01).The expression of TS had negative correlation with the antitumor effect of fluorouracil.The five-year survival rate of TS positive patients was lower than that of TS negative patients (P＜0.01).Conclusions Breast cancer patients with low expression of TS may benefit from fluorouracil.TS can be used as a molecule marker for predicting efficacy of breast cancer chemotherapy.

The Uneven Distribution of Mating Type Genes in Natural and Cultivated Truffle Orchards Contributes to the Fructification of Tuber indicum

The aim of this study was to investigate the pattern of distribution of mating type (MAT) genes of Tuber indicum in ectomycorhizosphere soils from natural T. indicum-producing areas and cultivated truffle orchards and ascocarp samples from different regions. Quantitative real-time PCR and multiplex PCR were used to weight the copy numbers of MAT1-1-1 and MAT1-2-1 in natural truffle soils and cultivated orchard soils. The effect of limestone on the pattern of truffle MAT genes and the correlation between soil properties and the proportion of MAT genes were also assessed. These results indicated that an uneven and nonrandom distribution of MAT genes was common in truffle-producing areas, cultivated truffle orchards, and ascocarps gleba. The competition between the two mating type genes and the expansion of unbalanced distribution was found to be closely related to truffle fructification. Limestone treatments failed to alter the proportion of the two mating type genes in the soil. The content of available phosphorus in soil was significantly correlated with the value of MAT1-1-1/MAT1-2-1 in cultivated and natural ectomycorhizosphere soils. The application of real-time quantitative PCR can provide reference for monitoring the dynamic changes of mating type genes in soil. This study investigates the distributional pattern of T. indicum MAT genes in the ectomycorhizosphere soil and ascocarp gleba from different regions, which may provide a foundation for the cultivation of T. indicum.

Molecular response to imatinib mesylate of Brazilian patients with chronic myeloid leukemia

Abstract Background Imatinib mesylate has revolutionized the treatment of chronic myeloid leukemia leading to significant reductions of BCR-ABL1 transcript levels in peripheral blood. Objective To evaluate the response to imatinib mesylate treatment (400 mg/day) in Brazilian patients in the chronic phase of chronic myeloid leukemia monitored by quantitative real time polymerase chain reaction. Methods Between October 2002 and October 2010, 3169 peripheral blood samples were collected from 1403 patients from 3 to 5 months, 6 to 11 months, 12 to 17 months, 18 to 23 months and ≥24 months after beginning imatinib treatment. Eighty-two patients had samples available and analyzed for all time intervals. BCR-ABL1 quantification was performed by quantitative real time polymerase chain reaction using the ABL1 gene as the control. Results of the BCR-ABL1 ratio as a percentage were reported by the international scale (IS) using the laboratory conversion factor (0.51). Results In the first interval, 80.8% of patients achieved the optimal response (BCR-ABL1 IS ≤ 10%). In the second period, 69.1% achieved optimal response (BCR-ABL1 IS ≤ 1%) and, between 12 and 17 months, 47.3% achieved major molecular response (BCR-ABL1 IS ≤ 0.1%). Conclusions The results of this retrospective study show that the response to imatinib treatment (400 mg/day) of Brazilian patients in the chronic phase of chronic myeloid leukemia is within the expected profile when compared to patients reported in international prospective randomized studies.

Benznidazole affects expression of Th1, Th17 and Treg cytokines during acute experimental Trypanosoma cruzi infection

Background The present study evaluated the effect of treatment with benznidazole on mRNA expression of IFN-γ, IL-17, IL-10, TGF-β and FoxP3 in spleen and heart tissue of BALB/c mice in the acute phase of an experimental infection with Trypanosoma cruzi, strains JLP or Y. Methods The mRNA expression of cytokines and parasite load were assessed by q-PCR. Dependent groups were compared using Student's paired t-test and independent groups were compared using Student's unpaired t-test. Results Infection with the JLP or Y strains increased expression of IFN-γ in the heart and of IL-10 and IL-17 in the spleen and heart compared to uninfected animals. Treatment increased the expression of IFN-γ and decreased the expression of IL-17, IL-10, TGF- β and Foxp3 in spleen and heart tissue compared to untreated infected animals. Conclusion Benznidazole can induce Th1 profile in the initial of the acute phase. The treatment decreased the parasite load in both organs, although the number of parasites in Y-strain-infected mice remained high. The data suggest that benznidazole may modulate cytokine expression in infection and can be dependent of the strain. However, treatment was not fully effective in the infection provoked by Y strain, probably due to the characteristics of the strain itself.(AU)

Benznidazole affects expression of Th1, Th17 and Treg cytokines during acute experimental Trypanosoma cruzi infection

Abstract Background The present study evaluated the effect of treatment with benznidazole on mRNA expression of IFN-, IL-17, IL-10, TGF- and FoxP3 in spleen and heart tissue of BALB/c mice in the acute phase of an experimental infection with Trypanosoma cruzi, strains JLP or Y. Methods The mRNA expression of cytokines and parasite load were assessed by q-PCR. Dependent groups were compared using Student's paired t-test and independent groups were compared using Student's unpaired t-test. Results Infection with the JLP or Y strains increased expression of IFN- in the heart and of IL-10 and IL-17 in the spleen and heart compared to uninfected animals. Treatment increased the expression of IFN- and decreased the expression of IL-17, IL-10, TGF- and Foxp3 in spleen and heart tissue compared to untreated infected animals. Conclusion Benznidazole can induce Th1 profile in the initial of the acute phase. The treatment decreased the parasite load in both organs, although the number of parasites in Y-strain-infected mice remained high. The data suggest that benznidazole may modulate cytokine expression in infection and can be dependent of the strain. However, treatment was not fully effective in the infection provoked by Y strain, probably due to the characteristics of the strain itself.

Determination of residual host cell DNA in recombinant human interferonα2 b substances by quantitative PCR / 中国生化药物杂志

Objective To develop and verify a method for determination of residual host cell DNA in recombinant human interferon α2b substances, which is used for the quality control of the product.Methods The residual host cell DNA was extracted by wako DNA extractor kit and determined by SYBRGreen based q-PCR using standard DNA as control.The residual host cell DNA was analyzed according to the standard curve.The developed method was verified by primer specifity, results accuracy and precision and used for determination of 3 batches of interferon substances. Results The minimum quantitative limit of residual host cell DNA by the developed method was 12 fg/μL, while the linear range was 12 fg/μL-120 ng/μL, with a correlation coefficient (r) of 0.998.The designed primers were specific to the DNA templates.The recovery rates of spiked samples with different DNA quantity were between 50%-200%.The residual host cell DNA determined by this method were not more than the limit, which were complied with the requirements for residual host cell DNA in Chinese Pharmacopeia ( volume III,2010 edition and 2015 edition) .Conclusion The wako DNA extractor kit could successfully solved the technical difficulties of sample pretreatment during residual DNA assay.The q-PCR method was simple, rapid and accurate for quantitation of residual host cell DNA in interferon substances.

ERCC1 expression in advanced breast cancer and its relationship with cisplatin resistance / 中华内分泌外科杂志

Objective To investigate ERCC1 expression in advanced breast cancer and its relationship with cisplatin resistance. Methods ERCC1 expression in 50 cases of advanced breast cancer was measured by RT-QPCR. The chemotherapy (CAF) was given. Gemcitabine and cisplatin were given because of metastasis. Results The expression of ERCC1 has no relationship with age, lymph node metastasis, clinicl stage, histologi-cal grade or human epidermal growth factor receptor-2 (HER2) expression. The effective rate was 18.8% (3/16) for ERCC1 high-expressed patients and 79.4%(27/34)for ERCC1 low-expressed patients in terms of cisplatin chemotherapy. The sensitity for cisplatin chemotherapy was high for patients with low ERCC1 expression and it was low for patients with high ERCC1 expression. The effective rate (complete remission+partial remission), and ineffective rate (stable disease+progressive disease) between the two had statastical significance (P<0.001). Con-clusions ERCC1 low-expressed patients can benefit from cisplatin. ERCC1 can be used as a moleculer marker for predicting chemotherapy efficacy in breast cancer.

Effects of aqueous extract of Cassiae Semen on activity hepatic microsomal CYP450 isozymes in rats / 中国中药杂志

To study the effect of aqueous extract of Cassiae Semen on the activity, mRNA and protein expressions of cytochrome P450(CYP450) system in rat liver microsomes, microsomes of rat liver were prepared after the oral administration with aqueous extract of Cassiae Semen for 14 days. The enzyme activity was quantified by Cocktail method. Meanwhile, the mRNA and protein expressions of CYP1A2, CYP2B1, CYP2C11, CYP2D2, CYP2E1 and CYP3A1 in the livers were detected by RT-PCR and Western blot. The result of this experiment was that aqueous extract of Cassiae Semen obviously induced the enzyme activities of CYP1A2, CYP2B1, CYP2C11, CYP2D2, CYP2E1 and CYP3A1. Low dose of aqueous extract of Cassiae Semen significantly reduced the activity of CYP2D2, but the activity of CYP2D2 was significantly induced by middle dose and high dose of aqueous extract of Cassiae Semen. These subtypes were increased in a dose-dependent manner except for CYP3A1. The mRNA levels of CYP1A2, CYP2C11, CYP2D2 and CYP2E1 were also induced in rats treated with aqueous extract of Cassiae Semen, but with no significant effect in CYP2B1 and CYP3A1 mRNA expressions. The protein levels of CYP2C11 and CYP2E1 were also induced in rats treated with aqueous extract of Cassiae Semen, but with no significant difference. Since the enzyme activity, mRNA and protein expressions of CYP450, particularly CYP2C11and2E1subtypes, were induced or inhibited by aqueous extract of Cassiae Semen to varing degrees, suggesting the potential drug-drug interactions should be concerned.

Expression levels of BDNF, trkB and ChAT in the brain of adult tree shrew (Tupaia belangeri) / 中国比较医学杂志

Objective To investigate the expression levels of BDNF, trkB and ChAT mRNA and proteins in the brain of adult tree shrews ( Tupaia belangeri ) .Methods Quantitative real-time PCR was employed to detect the expression levels of BDNF, trkB and ChAT mRNA in the hippocampus, basal ganglia and frontal cortex of adult tree shrews.The expression levels of BDNF, trkB and ChAT proteins andβ-actin was used as internal standard.Results The expression level of BDNF mRNA was highest in the hippocampus of adult tree shrew, and there were significant differences between that in the hippocampus, and basal ganglia and frontal cortex (P0.05) in the expressions of trkB protein among the hippocampus, basal ganglia and frontal cortex of the adult tree shrews.There were no significant differences in expressions of ChAT mRNA and protein among the hippocampus, basal ganglia and frontal cortex in adult tree shrews ( P>0.05 ) . Conclusions The expression levels of ChAT mRNA were consistent with that of ChAT protein in the hippocampus, basal ganglia and frontal cortex of adult tree shrews, while the expression levels of BDNF and trkB mRNA were not consistent with their proteins, which might indicate that the transcriptional regulation pattern might be more complex.Tree shrew is a valuable animal model in the study of mechanism of BDNF/trkB gene expression.

Prediction of breast cancer and prognosis by 21-gene oncotype Dx / 中华内分泌外科杂志

Objective To study the prognostic and predictive significance of 21-gene assay ( oncotype DX)in breast cancer.Methods Real-time quantitative PCR(RT-QPCR)was used to detect 21 gene expression in breast cancer tissues (100 cases)and recurrence score(RS)was calculated.Results Among the 100 cases, 52 cases had low RS , 22 cases had middle RS , and 26 cases had high RS .The recurrence rate of five years was 1.92%,4.55%and 15.38%respectively.21 gene expression had nothing to do with patients'age, tumor size, histological grade , lymph node metastasis state , ER expression , or PR expression .It was associated with HER 2 expression .Conclusions 21 genes is a good prediction factor in breast cancer and its prognosis .

Apoptotic effect of HBO exposure on cerebral vascular endothelial cells in rabbits / 重庆医学

Objective To investigate the effects of hyperbaric Oxygen (HBO)exposure on Caspase-3 and Bcl-2 mRNA ex-pressions in both internal carotid artery (ICA)and basal artery (BA)in rabbits.Methods Twenty-four healthy adult New Zealand rabbits were randomly divided into 2 groups:HBO group and the control group,with each group consisted of 12 animals.The rab-bits in the HBO group were exposed to HBO at 2.2 ATA for 60 minutes each day for 3 successive days.The rabbits in the control group were normally fed without any treatment.Real-time PCR was used to detect Caspase-3 and Bcl-2 mRNA expressions in both ICA and BA in 2 groups.Results HBO significantly decreased the Caspase-3 mRNA expression [(0.038 ±0.006 )vs .(1.000 ± 0.225)]and increased the Bcl-2 mRNA expression [(1.877 ±0.1 69)vs .(1.000 ±0.364)].In ICA,HBO similarly decreased the Caspase-3 mRNA expression [(0.41 9±0.091)vs .(1.000 ±0.1 75)]and increased the Bcl-2 mRNA expression [(1.269 ±0.270) vs .(1.000±0.1 1 7)]in BA.All the differences mentioned above were of statistical significance (P <0.01).Conclusion HBO ex-erts an inhibition effect on apoptosis in cerebral vascular endothelial cells.The mechanism may be related to inhibiting the expres-sion of Caspase-3 mRNA and promoting the expression of Bcl-2 mRNA.

Combination of duplex PCR technology with capillary electrophoresis is an alterna-tive method to validate the result of transcriptome sequencing / 军事医学

Objective To establish a method to validate the result of transcriptome sequencing using duplex PCR tech -nology combined with capillary electrophoresis .Methods According to a previous study on transcriptome sequencing , eight differentially expressed genes were chosen as target genes for examination .The mRNA expression level of these genes was detected using duplex PCR combined with agarose gel electrophoresis , duplex PCR combined with capillary electropho-resis and Q-PCR, respectively.Then, the verification efficiency of each method was evaluated carefully .Results The ver-ification efficiency of duplex PCR combined with agarose gel electrophoresis was 50%, while that of duplex PCR combined with capillary electrophoresis and Q-PCR was both 100%.Conclusion Combination of duplex PCR technology with capil-lary electrophoresis can be used as an alternative method to validate the results of transcriptome sequencing .

Effects of Shenmai injection on the regulation of heart CYP450 isozymes in rats / 中国药理学通报

Aim To investigate the influence of Shen-mai injection ( SMI ) on the expression of cytochrome P450(CYP450) system in rat′s hearts. Methods Rat hearts were prepared after a fourteen-day continuous administration of SMI. The expression of several CYP genes, ANP, BNP and EPHX2 were measured by qPCR. Results SMI induced the increase in the ex-pression of other CYP genes except CYP2 B1、CYP4 A3 and CYP4 F6;HSI caused an induction of CYP2 E1 , CYP4A3,CYP4F1 and EHPX2 as compared with the control. In addition, there was a significant induction of ANP, BNP and EHPX2 and a significant inhibition of CYP2B1 and CYP2C11 after treated with MDI. Conclusion Although there is no significant change in the gene expression of CYP2 B1 after the treatment with SMI, but there is a general trend of induction, and MDI shows a significant inhibition of CYP2 B1 , therefore HSI has greater effect on CYP 2 B 1 than MDI . SMI causes a significant induction of CYP2 E1 , CYP4F1 and EHPX2 , similarly there is an induction of CYP2E1,CYP4F1 and EHPX2 by HSI and MDI, indi-cating that Hongshen and Maidong are both involved in the induction. MDI has a greater inductive effect than HSI on ANP and BNP. SMI is widely used for the treatment of cardiovascular diseases due to its regula-tion of CYP2J3、ANP and BNP mRNA expression.

Detection of Acute Toxoplasmosis in Pigs Using Loop-Mediated Isothermal Amplification and Quantitative PCR

A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods.

Quantitative determination of adenovirus and carmustine in anionic liposomes-mediated co-delivery system / 中国药学杂志

OBJECTIVE: To establish effective and reliable methods for the determination of adenovirus and carmustine in anionic liposomes-mediated co-delivery system. METHODS: The complexes of co-delivery system were prepared by calcium-induced phase changing method. For carrying out the quantitative-PCR (Q-PCR) detection, the sequences of primer and probe were designed accoding to the hexon gene sequences of Ad5, and then the reaction system of Q-PCR was optimized to detect the loading rate of Ad5. The HPLC condition was also optimized to determine the drug loading of carmustine in the co-delivery system. RESULTS: In the co-delivery system, the loading rate of adenovirus detected by Q-PCR method was (23.2±1.8)% and that of carmustine was (55±2.8)%. CONCLUSION Both of Q-PCR and HPLC methods have been successfully used for the quantification of adenovirus and carmustine in anionic liposomes-mediated co-delivery system. These methods are simple, reliable and accurate, which can be used in other similar experiments.

PML-RARalpha gene detection method optimization for quantitative PCR

Hybrid gene PML-RARα is the molecular target found in most cases of acute promyelocytic leukemia (APL) and has been used for diagnosis and minimal residual disease studies. The standard molecular technique employed is qualitative reverse transcriptase-polymerase chain reaction (RT-PCR), but with the emergence of real time PCR (Q-PCR), PML-RARα gene detection approaches have been described allowing transcript detection, with the methodological advantage of eliminating post-PCR processing. However, current protocols report the use of expensive fluorescent labeled probes, limiting its routine application in the laboratory. The objective of this study was to optimize PML-RARalpha gene detection method for Q-PCR, using SYBR® Green fluorescent dye. The analysis was performed with NB4 cellular lineage cDNA. Thermal cycling protocols, cDNA synthesis with random or specific primer and different MgCl2 and amplification primers concentrations were tested. Results show that amplification improved in the following conditions: 2 mM MgCl2, 10 pmol primers and cDNA synthesized with specific primer. There were no significant differences using annealing temperature (58ºC/30 s) followed by extension (72ºC/30 s) or annealing associated with extension as a single step (60ºC/45 s). This paper demonstrates the optimization of PML-RARα gene detection for Q-PCR studies using a technique considered sensitive and less expensive for routine use in the laboratory.

O gene híbrido PML-RARα é o marcador molecular presente na maioria dos casos de leucemia aguda promielocítica (LAP), sendo útil ao diagnóstico e ao estudo da doença residual mínima. A técnica molecular empregada como rotina laboratorial é a reação em cadeia da polimerase com transcrição reversa (RT-PCR) qualitativa, porém com o surgimento da PCR em tempo real (Q-PCR), foram descritas abordagens de detecção do gene PML-RARalfa possibilitando a quantificação de transcritos, com a vantagem metodológica da eliminação do processamento pós-PCR. No entanto, os protocolos relatam o uso de sondas fluorescentes de custo elevado para a rotina clínica, limitando sua aplicação. Este estudo teve como objetivo otimizar o método de detecção do gene PML-RARα para Q-PCR, utilizando como sistema de marcação fluorescente o intercalante SYBR® Green. A análise foi realizada com cDNA da linhagem celular NB4, tendo sido testados protocolos de termociclagem, síntese de cDNA com primer randômico ou específico e diferentes concentrações de MgCl2 e primers para amplificação. Os resultados mostraram amplificação mais eficiente nas seguintes condições: 2 mM MgCl2, 10 pmol de primers e cDNA sintetizado com primer específico. Não houve diferença na utilização de etapas para anelamento (58ºC/30 s) seguido de extensão (72ºC/30 s) ou etapa única de anelamento associado à extensão (60ºC/45 s). Esses resultados demonstram a otimização da detecção do gene PML-RARα para Q-PCR através de um método considerado sensível e de baixo custo para a rotina laboratorial.