Fluorescent Reverse Transcription-polymerase Chain Reaction Research Articles

Evaluation of enterovirus nucleic acids detection method based on ultra‐fast real‐time fluorescence RT‐PCR technology—A pilot study

The outbreak of COVID-19 epidemic has enabled the establishment and application of various rapid detection methods. It is particularly important to establish a fast and accurate detection method for enterovirus, which will be beneficial for clinical diagnosis, epidemic prevention and control, and timely traceability. Through establishing an ultra-fast reverse transcription-polymerase chain reaction (RT-PCR) equipment, this study aimed to evaluate the sensitivity and specificity of the testing method of enterovirus nucleic acids based on ultra-fast real-time fluorescence RT-PCR technology. A total of 61 cases were sampled, which were then transported and preserved. After the nucleic acid extraction, the nucleic acids of the same sample were tested with the enterovirus nucleic acid detection kit produced by Guangzhou Da An Gene Company and the ultra-fast RT-PCR equipment system established in this study. ABI7500Fast and Ahram biosystems S1 fast equipment were used for amplification detection. If the sample had an S-shaped amplification curve in the FAM channel and the Ct value ≤40.00, the result was positive. The sensitivity, precision, and accuracy of the detection method were then verified. This study established a novel testing method to achieve enterovirus nucleic acid detection within 24 min. The sensitivity detection limit of the method was 1.0 × 102 copies/ml. The coefficients of variation for repeated detection of the high, medium, and low concentration samples were 2.644%, 1.674%, and 4.281%, respectively, with good detection repeatability. In addition, a total of 29 cases were positive by the ultra-fast RT-PCR detection method in 61 suspected samples, which was consistent with the conventional fluorescent RT-PCR method. The established rapid detection method can greatly shorten the time for providing a detection report, which may greatly improve the efficiency of diagnosis and treatment.

Results analysis on SARS-CoV-2 nucleic acid detection based on different target gene detection

Objective To compare the results of three SARS-CoV-2 nucleic acid detection reagents (reagent A, B and based on different target gene detection, and we analyze the consistency by preliminary evaluation of the application of each reagent. Methods Totally 43 samples of pharyngeal swabs were collected from our hospital and the designated treatment hospital, and detected by the real-time fluorescencereverse transcription-polymerase chain reaction (RT-PCR) in three SARS- CoV-2 nucleic acid detection reagent (reagent A, B and C) at the same time. Clinical diagnosis was divided into COVID-19 group ( n =31) and control group ( n =12). Fisher exact probability test and Kendall coordination coefficient W test were used to test the difference of positive rate and consistency of test results. Results The positive detection rate of reagent B was the highest at 90.32%, and there was no reexamination result.The positive detection rate of reagent A and reagent C were 77.42% and 74.19%, and more reexamination results were needed for reagent C. In the control group, reagent A and C did not test positive, but reagent B showed one positive result. The difference of positive detection rate among reagents was not statistically significant ( P >0.05), and the difference in consistency was statistically significant, with progressive significance Sig=0.00 and Kendall’s W coefficient value being 0.852, indicating that the consistency of test results of the three reagents was very high. Conclusion The three reagents showed good consistency in the nucleic acid test results for COVID-19. Reagent B has advantages in sensitivity, and reagent A and C are stable in specificity.Reagent B is most suitable for strict screening during an outbreak, but false positives should be noted. Reagent A and reagent C need to improve the quality of test samples for false negative and reexamination results, and the result interpretation method of reagent C is not good in the processing of large quantities of positive sample data. 摘要： 目的 比较三种不同靶基因检测 2019 新型冠状病毒(SARS-CoV-2)的检测结果，分析其一致性，初步评估各 试剂的应用情况。 方法 收集 43 份本院患者和定点治疗医院送来的咽拭子标本，根据临床诊断分为新型冠状病毒肺 炎(COVID-19)组 31 份和对照组 12 份，用三种 SARS-CoV-2 核酸检测试剂同时进行实时荧光逆转录聚合酶链反应(re⁃ verse transcription-polymerase chain reaction, RT-PCR)检测，检测结果的阳性率差异和一致性分别用 Fisher 精确概率检 验和 Kendall’s 协调系数 W 检验。 结果 在 COVID-19 组中试剂 B 的阳性检出率最高为 90.32%，且不存在需复检结果； 试剂 A 和试剂 C 的阳性检出率分别为 77.42% 和 74.19%，且试剂 C 出现的需复检结果较多；在对照组中试剂 A 和试剂 C 未检测出阳性，但试剂 B 出现一例阳性结果。COVID-19 组中各试剂的阳性检出率差异不具有统计学意义( P >0.05)；一 致性方面差异具有统计学意义，渐进显著性 Sig=0.00，Kendall’sW 系数值为 0.852，说明三种试剂检测结果的一致性程度 很高。 结论 三种试剂对 COVID-19 核酸检测的结果一致性很好，灵敏度方面试剂 B 较有优势，特异性方面试剂 A 和 试剂 C 较稳定。试剂 B 最适合疫情期间的严格排查，但应注意假阳性结果的出现；试剂 A 和试剂 C 针对假阴性和需复 检结果需要提高检测样本的质量，且试剂 C 的结果判读方式在处理大批量阳性标本数据时体验欠佳。

Discrimination of False Negative Results in RT-PCR Detection of SARS-CoV-2 RNAs in Clinical Specimens by Using an Internal Reference

Reverse transcription-polymerase chain reaction (RT-PCR) is an essential method for specific diagnosis of SARS-CoV-2 infection. Unfortunately, false negative test results are often reported. In this study, we attempted to determine the principal causes leading to false negative results of RT-PCR detection of SARS-CoV-2 RNAs in respiratory tract specimens. Multiple sputum and throat swab specimens from 161 confirmed COVID-19 patients were tested with a commercial fluorescent RT-PCR kit targeting the ORF1ab and N regions of SARS-CoV-2 genome. The RNA level of a cellular housekeeping gene ribonuclease P/MRP subunit p30 (RPP30) in these specimens was also assessed by RT-PCR. Data for a total of 1052 samples were retrospectively re-analyzed and a strong association between positive results in SARS-CoV-2 RNA tests and high level of RPP30 RNA in respiratory tract specimens was revealed. By using the ROC-AUC analysis, we identified Ct cutoff values for RPP30 RT-PCR which predicted false negative results for SARS-CoV-2 RT-PCR with high sensitivity (95.03%–95.26%) and specificity (83.72%–98.55%) for respective combination of specimen type and amplification reaction. Using these Ct cutoff values, false negative results could be reliably identified. Therefore, the presence of cellular materials, likely infected host cells, are essential for correct SARS-CoV-2 RNA detection by RT-PCR in patient specimens. RPP30 could serve as an indicator for cellular content, or a surrogate indicator for specimen quality. In addition, our results demonstrated that false negativity accounted for a vast majority of contradicting results in SARS-CoV-2 RNA test by RT-PCR.Electronic supplementary materialThe online version of this article (10.1007/s12250-020-00273-8) contains supplementary material, which is available to authorized users.

Methods and significance of the combined detection of HER2 gene amplification and chemosensitivity in gastric cancer.

This study aims to investigate the significance of combined detection of HER2 gene amplification and chemosensitivity in gastric cancer. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) and fluorescence reverse-transcription polymerase chain reaction (RT-PCR) were used to analyze the expression of HER2 protein, HER2 gene amplification and the mRNA expression of ERCC1, TUBB3 and TYMS genes in 135 cases of gastric carcinoma. The expression rate of HER2 protein was 39.3% (53/135). Among these positive cases, patients with HER2 protein (3+) accounted for 9.6% (13/135), patients with HER2 protein (2+) accounted for 13.3% (18/135), and patients with HER2 protein (1+) accounted for 16.3% (22/135). The amplification rate of the HER2 gene was 35.8% (19/53). In the detection of the mRNA expression of ERCC1, TUBB3 and TYMS, 45 patients had low and moderate single gene expression, 50 patients had low and moderate double gene expression, 22 patients had low and moderate mRNA expression for ERCC1, TUBB3 and TYMS, and 18 patients had no low and moderate expression. Among the 53 patients with HER2 protein expression and 22 patients with low and moderate mRNA expression of ERCC1, TUBB3 and TYMS, 12 patients received chemotherapy and trastuzumab. Follow-up results revealed that HER2 gene status was positively correlated with the therapeutic effect of the combined treatment in patients with low mRNA expression of ERCC1, TUBB3 and TYMS. Among these patients, five patients with extensive HER2 (3+), HER2 cluster-specific amplification, and low mRNA expression of ERCC1, TUBB3 and TYMS had a total survival of up to 19.1 months. The detection of HER2 in gastric cancer is highly heterogeneity, and the combined detection of HER2 protein expression, HER2 gene amplification and chemosensitivity can provide important reference markers for the benefit of antitumor drugs.

Effect of multiple cycles of freeze-thawing on the RNA quality of lung cancer tissues.

RNA degradation is a major problem in tissue banking. We explored the effect of thawing flash-frozen biospecimens on the quality and integrity of RNA for genetic testing as well as for other cancer research studies. The histological quality of the frozen tumor sections was evaluated by using hematoxylin and eosin staining. RNA extraction from 60 lung cancer tissue samples subjected to various freeze/thaw cycles was performed using the RNeasy Plus isolation kit. RNA integrity was assessed by using an Agilent bioanalyzer to obtain RNA integrity numbers (RIN). Furthermore, RNA from different groups was used for fluorescence Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene mutation to verify whether it can be used for research or clinical testing. Highly variable RIN values were observed among the samples, which showed no correlation with the number of freeze/thaw cycles conducted. However, after 3 freeze/thaw cycles (each thaw event lasted for 10min), an increasing number of changes in peak intensity in RINs were observed. After 5 freeze/thaw cycles, RNA integrity decreased to approximately 35%. After 3 freeze/thaw cycles, the RNA could still be used for RT-PCR analysis of EML4-ALK fusion gene mutations; whereas those subjected to 5 freeze/thaw cycles could not. Limited (<3) freeze/thaw cycles did not adversely affect the quality of RNA extracted from tumor tissues and subsequent RT-PCR analysis. Our data could be utilized in the establishment of a standardized procedure for tissue biospecimen collection and storage.

Effect of down-regulation of high mobility group box-1 expression on proliferation and migration abilities of human umbilical vein endothelial cells

To investigate the effect of down-regulation of high mobility group box-1 (HMGB1) expression on the proliferation and migration abilities of human umbilical vein endothelial cell ( HUVEC) in vitro. The method of stable transfection was used to transfect the pRNA-u6.1/Neo-control and pRNA-u6.1/ Neo-HMGB1 short hairpin RNA (shRNA) plasmid to HUVEC cells, and control and HMGB1 shRNA cell lines were reproduced, and the cells were identified by Western blotting test and real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR). Then methyl thiazolyl tetrazolium (MTT) was used for the determination of the proliferative ability, flow cytometry was used for determining the changes in cell cycle distribution ability of HUVEC, and the migratory ability was detected by scratch test. Western blotting and RT -PCR detection proved the production of stable transfection cell lines was successful, and the content of HMGB1 protein and mRNA expression in cells were significantly lower after cultivation for 72 hours than those of control cells (protein: 0.436 ± 0.027 vs. 1.017 ± 0.038, I= 12.180, P=0.000; mRNA: 0.436 ± 0.031 vs. 1.020 ± 0.051, T=9.660, P= 0.001 ). The results of MTT showed that the proliferation ability of HMGB1 shRNA cell lines was lower obviously 2, 3, 4, 5 days after transfection than that of control cells ( 2 days: 0.210 ± 0.023 vs. 0.240 ± 0.011, T= 1.050, P=0.351; 3 days: 0.240 ± 0.022 vs. 0.361 ± 0.030, T=3.203, P=0.033; 4 days: 0.373 ± 0.031 vs. 0.531 ± 0.033, T=3.530, P=0.022; 5 days: 0.441 ± 0.031 vs. 0.602 ± 0.030, T=4.180, P=0.106). Flow cytometry results showed that the number of HMGB 1 shRNA cells in S phase was significantly lower than that of the control cell line ( ( 13.10 ± 1.10 )% vs. (21.12 ± 1.20)%, T=4.950, P=0.001). Scratch test showed that the healing ability of HMGB1 shRNA cell line was lowered significantly at 12 hours as compared with that of control ( (20.17 ± 3.33 )% vs. ( 88.53 ± 3.15 )% , T= 14.142, P=0.000). HMGB 1 shRNA can significantly inhibit HUVEC cell proliferation and migration.

Selection of housekeeping genes in rat model of myocardial infarction

Objective Reverse transcription-polymerase chain reaction (RT-PCR) is widely used in gene expression analysis.Selection of proper housekeeping gene is very important.Rat model of myocardial infarction is a major animal model of myocardial infarction.Few reports are found about the selection of rat housekeeping gene after myocardial infarction.This study was aimed to explore the stability of the housekeeping genes expression in the rat model of myocardial infarction.Methods Myocardial infarction models were made using the anterior descending coronary artery ligation method.Combining with practical work,analysis and selection were made of four widely used standard housekeeping genes:glyceraldehydes-3-phosphate dehydrogenase (GAPDH),ribosomal protein L13A (RPL13A),beta-actin (ACTB) and acidic ribosomal phosphoprotein P0 (ARBP).The expressions in the rat heart were compared using real-time fluorescent RT-PCR and analysis was carried out to figure out which was the most suitable housekeeping gene for study of gene expression in rats after myocardial infarction using GeNorm program.Results The M values of RPL13A,GAPDH,ARBP and ACTB gene were 0.812,0.721,0.812 and 1.2 respectively.Conclusion GAPDH and ARBP are the most stable genes for the rat myocardial infarction model. Key words: Housekeeping gene; GeNorm program; Gene expression

Isolation and sequence analysis on virus of HFMD with encephalitis in Yantal city

To learn about the pathogen spectrum and genetic characterization of HFMD with encephalitis in Yantai city. Stool samples and cerebrospinal fluids (CSF) collected from HFMD with encephalitis cases in Yantai. Virus were isolated from stool samples and identified by fluorescence reverse transcription-polymerase chain reaction. The VPl region was amplified and sequenced from positive specimens. Genetic characterization was identified by sequence analysis. Getting 3 virus strains from 10 stool specimens and all of them belong to EV71. The nucleotide and amino acid homogeneity with the representative isolates of C4a were 98%-99% and 98.90%-99.45% respectively. The pathogen of HFMD with encephalitis in Yantai city were mainly EV71 wich belong to subgenogroup C4 cluster C4a.

Matrix Metalloproteinase‐1 of Gingival Fibroblasts Influenced by Advanced Glycation End Products (AGEs) and Their Association With Receptor for AGEs and Nuclear Factor‐κB in Gingival Connective Tissue

The effect of advanced glycation end products (AGEs) on gingival inflammation has not been fully elucidated. This study aims to investigate the hypothesis that AGEs may enhance the expression of matrix metalloproteinase-1 (MMP-1) of human gingival fibroblasts (HGFs) and to explore whether the signal pathway receptor for AGE (RAGE)/nuclear factor-κB (NF-κB) are involved in the expression of MMP-1 in HGFs. Cultured HGFs from 12 healthy gingival human tissue samples were coincubated with AGEs for the detection of MMP-1 protein and mRNA. Thirty-six gingival samples were collected and treated for the determination of RAGE, NF-κB, and MMP-1 mRNA level in gingival connective tissues from the participants with chronic periodontitis, diabetes-associated periodontitis, and healthy controls. Enzyme-linked immunosorbent assay and real-time fluorescence reverse transcription-polymerase chain reaction were used for the measurement of protein and mRNA level, respectively. In addition, clinical periodontal parameters were also checked. AGEs strongly induced MMP-1 mRNA and protein expression in HGFs and in a time- and concentration-dependent manner (P <0.05). In gingival connective tissue, the level of both RAGE mRNA and NF-κB mRNA were higher in patients with periodontitis than in healthy controls (P <0.05). There was significant correlation between the level of RAGE mRNA and NF-κB mRNA (R(2) = 0.90, P <0.05). Accumulation of AGEs may upregulate the expression of MMP-1 by HGFs, which may play a role in the development of diabetes-associated periodontitis, and RAGE/NF-κB pathway may be involved in metabolism of MMP-1 in HGFs.

Molecular epidemiology of human enterovirus 71 strains in Qingdao region, Shandong province, 2007 - 2009

To study the molecular epidemiology of human enterovirus 71 (EV71) isolated from patients with hand-foot-mouth disease (HFMD) in Qingdao region between 2007 and 2009. Throat swabs of HFMD were detected for total enterovirus (EV), EV71 and CA16 by fluorescence reverse transcription-polymerase chain reaction (RT-PCR). The VP1 region was amplified from positive EV71 specimens and 51 nucleotide sequences of VP1 gene were successfully sequenced. EV71 genotypes were characterized by phylogenetic analysis. 51 Qingdao EV71 strains identified between 2007 and 2009 belonged to C4a cluster of subgenotype C4 and 5.3% nucleotide divergence and 1.7% amino acid divergence were found among them. The strains in 2008 and 2009 were respectively divided into several lineages on phylogenetic trees but there was a main lineage (namely prominent strain) every year. The Qingdao prominent strains identified in 2008 had high identities with the Linyi prominent strains in 2007. The Qingdao prominent strains in 2009 and the secondary prominent strains in 2008 had high identities with the Fuyang prominent strains in 2008. Two Qingdao strains in 2007 had high identities with the Linyi popular strains in 2007. Data showed that several EV71 transmission chains were co-circulating in Qingdao between 2008 and 2009. The main transmission chain in 2008 became the minor one but the minor transmission chain in 2008 was transferred into the main transmission chain in 2009.

Effects of combined siRNA-TR and -TERT on telomerase activity and growth of bladder transitional cell cancer BIU-87 cells

The effects of combined RNA interference (RNAi) of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) genes on telomerase activity in a bladder cancer cell line (BIU-87 cells) were investigated by using gene chip technology in vitro with an attempt to evaluate the role of RNAi in the gene therapy of bladder transitional cell cancer (BTCC). Three TR-specific double-stranded small interfering RNAs (siRNAs) and three TERT-specific double-stranded siRNAs were designed to target different regions of TR and TERT mRNA. The phTR-siRNA, phTERT-siRNA, and the combination of both plasmids phTR+phTERT-siRNA were transfected into BIU-87 cells. The expression of hTR and hTERT mRNA was detected by quantitative fluorescent reverse transcription-polymerase chain reaction, and a telomeric repeat amplification protocol was applied to detect telomerase activity. Growth inhibition of BIU-87 cells was measured by MTT assay. Gene chip analysis was performed to evaluate the effects of the combined RNAi of hTR+hTERT genes on telomerase activity and growth of BIU-87 cells in vitro. The results showed that the expression of hTERT and hTR mRNA was inhibited by pRNAT-hTERT-III, pRNAT-hTR-III, and pRNAT-hTR-III+hTERT-III in BIU-87 cells. The inhibition efficiency of pRNAT-hTERT-III, pRNAT-hTR-III, pRNAT-hTERT-III+pRNAT-hTR-III was 67% for TERT mRNA, 41% for TR mRNA, 57% for TR mRNA and 70% for TERT mRNA in BIU-87 cells respectively. The growth of BIU-87 cells was inhibited and telomerase activity was considerably decreased, especially in the cells treated with combined RNAi-hTR and -hTERT. Gene chip analysis revealed that 21 genes were down-regulated (ATM, BAX, BCL2, BCL2L1, BIRC5, CD44, CTNNB1, E2F1, JUN, MCAM, MTA1, MYC, NFKB1, NFKBIA, NME4, PNN, PNN, SERPINE1, THBS1, TNFRSF1A, and UCC1). The results indicated that hTR-siRNA and hTERT-siRNA, especially their combination, siRNA hTR+hTERT, specifically and effectively suppressed the expression of both hTR and hTERT mRNA and telomerase activity. Molecular biological mechanism by which combined siRNA-TR and -TERT inhibited telomerase activity and growth of BIU-87 cells in vitro may involve the down-regulation of the 21 genes.

A study on the mechanism of 5-aminolevulinic acid photodynamic therapy in vitro and in vivo in cervical cancer

5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is a new ablation treatment for tumors, while its function mechanism in cervical cancer has not been elucidated. In this study, we investigated the effects of ALA-PDT on the cervical cancer cell line Me180, to search for optimal parameters of PDT. ALA-PDT on proliferation of Me180 was examined by MTT assay to find the optimal function parameters of ALA-PDT. Apoptosis was observed by using AnnexinV-FITC/PI double staining, Hoechst 33342 staining and May-Grünwald-Farbstoff Giemsa staining. Furthermore, we established a tumor model and 6 mice of each group underwent measurement of the tumor size on days 3, 7, 14, and 21 after treatment. The mRNA expression of survivin, bcl-2, p53, bax and bad in Me180 cells were detected by real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR) in vitro and in vivo. Finally, we compared the effects between topical and intravenous administration. Based on the above studies, we found ALA-PDT induced apoptosis and G0-G1 phase arrest of Me180 cells. The tumor volume of the topical administration and PDT group was the smallest at 7-14 days post-PDT. H&E staining showed remarkable subcutaneous necrosis in the PDT groups. The mRNA expression of survivin and bcl-2 in Me180 cells were suppressed post-PDT. Topical administration of PDT is recommended in treating cervical cancer so as to minimize the side-effects and inconvenience of phototoxic reaction brought by PDT. Our data may contribute to making the mechanism of PDT on cervical cancer clearer and give some useful suggestions for clinical application.

A sensitive assay for measuring SMN mRNA levels in peripheral blood and in muscle samples of patients affected with spinal muscular atrophy

Different therapeutic strategies are currently evaluated in spinal muscular atrophy (SMA) that are aimed at increasing full-length (FL) mRNA levels produced from the SMN2 gene. Assays measuring SMN mRNA levels are needed. We have developed a sensitive, comparative assay based on multiplex fluorescent reverse-transcription polymerase chain reaction (RT-PCR) that can measure, in the same reaction, the levels of SMN mRNA with and without exon 7 sequences as well as those of total SMN mRNA. This assay allows to calculate directly the ratios of FL SMN mRNA to SMN mRNA without exon 7 (Delta7). We have used this assay to compare the levels of SMN transcripts in the blood of 75 unrelated normal subjects and of 48 SMA patients, and in muscle samples of 8 SMA patients. The SMN1 and the SMN2 genes produced very similar levels of total mRNA. Levels of transcripts lacking exon 7 were linearly dependent on the number of SMN2 copies, both in SMA patients and in controls. In patients, FL mRNA levels correlated with SMN2 copy number. A significant but weaker inverse correlation was also observed between FL or Delta7 mRNA levels and disease severity, but patients with three SMN2 copies and different SMA types displayed similar mRNA levels. A significantly higher FL to Delta7 ratio was measured in blood cells than in skeletal muscle (0.80+/-0.18 versus 0.47+/-0.11). This assay can be used as a sensitive biomarker for monitoring the effects of various drugs in forthcoming clinical trials of SMA.

Human Neutralizing Fab Molecules against Severe Acute Respiratory Syndrome Coronavirus Generated by Phage Display

Human recombinant Fab fragments specific for the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV) were screened from a human Fab library, which was generated from RNAs from peripheral lymphocytes of convalescent SARS patients. Among 50 randomly picked clones, 12 Fabs specially reacted with S protein by an enzyme-linked immunosorbent assay. The microneutralizing test showed that one clone, designated M1A, had neutralizing activity on Vero E6 cells against SARS-CoV. DNA sequence analysis indicated that the light- and heavy-chain genes of M1A Fab belong to the kappa2a and 4f families, respectively. A neutralizing test on purified M1A demonstrated that 0.5 mg/ml of M1A completely inhibited SARS-CoV activity, with an absence of cytopathic effect for 7 days. Real-time fluorescence reverse transcription-PCR also proved the neutralizing capacity of M1A. These data showed that the number of virus copies was significantly reduced in the M1A-treated group, suggesting an important role for M1A in passive immunoprophylaxis against the SARS virus.

Determination of murine fetal Cyp1a1 and 1b1 expression by real-time fluorescence reverse transcription-polymerase chain reaction

We describe the application of quantitative RT-PCR methodology to study expression of Cyp1a1 and 1b1 in murine fetal tissue. We applied real-time PCR using the fluorescent dye, SYBR Green, to establish a very reliable, sensitive, specific, inexpensive, and rapid assay that was linear over a wide dynamic range of starting target gene copy number. The main point of this paper is to provide a detailed description of how to optimize real-time reverse transcription-polymerase chain reaction (RT-PCR) assays and, most importantly, to provide solutions for some of the commonly observed problems encountered when employing quantitative RT-PCR methods. We establish that use of SYBR Green for detection of PCR products was a powerful and reliable tool in our investigation of Cyp gene expression in murine fetal tissues, and demonstrate the kinetics of induction of Cyp1a1 and 1b1 in crosses between C57BL/6 and Balb/c mice. In addition, we demonstrate for the first time the induction of Cyp1b1 RNA expression in fetal lung and liver tissues following in utero exposure to 3-methylcholanthrene (MC), a polycyclic aromatic hydrocarbon. This assay for Cyp1a1 and 1b1 expression could be utilized equally well to detect other genes of interest in a variety of toxicological investigations.

Specific detection and quantification of Plum pox virus by real-time fluorescent reverse transcription-PCR

Plum pox virus (PPV), a destructive and economically devastating pathogen of Prunus species, was recently discovered in Pennsylvania and Canada. Current containment efforts involve eradication of infected trees based on ELISA surveys, which are laborious and less sensitive than PCR-based techniques. A real-time, fluorescent, reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of PPV in the Smart Cycler (Cepheid). The methods developed are reproducible, specific to PPV, and sensitive enough to consistently detect PPV transcripts at the 10–20 fg level. The assay is more sensitive than either ELISA or traditional PCR followed by visualization with ethidium–bromide. PPV was detected from multiple hosts and from multiple Prunus tissues (leaf, stem, bud, and root). A dilution series using an in vitro synthesized transcript containing the target sequence as a standard demonstrated that the assay was effective for quantitation of viral template. The real-time PCR assay is a valuable tool for PPV detection and liter quantification in field or laboratory settings.

Teriparatide (Human Parathyroid Hormone (1–34)) Inhibits Osteogenic Vascular Calcification in Diabetic Low Density Lipoprotein Receptor-deficient Mice

Cardiovascular calcification is a common consequence of diabetes. High fat diets induce diabetes and arterial calcification in male low density lipoprotein receptor (LDLR) -/- mice; calcification occurs via Msx2 signaling that promotes the osteogenic differentiation of arterial myofibroblasts. We studied regulation of arterial osteogenesis by human parathyroid hormone (PTH) (1-34) (also called teriparatide) in LDLR -/- mice fed diabetogenic diets for 4 weeks. LDLR -/- mice were treated with vehicle or 0.4 mg/kg of PTH(1-34) subcutaneously five times/week. Gene expression was determined from single aortas and hind limb RNA by fluorescence reverse transcription-PCR. Valve calcification was determined by histological staining of cardiac sections using image analysis to quantify valve leaflet mineralization. PTH(1-34) increased bone mineral content (by dual energy x-ray absorptiometry) in LDLR -/- mice, with induction of osseous osteopontin (OPN) expression and serum OPN levels (>150 nM); PTH(1-34) did not significantly change serum glucose, lipids, body weight, or fat mass. PTH(1-34) suppressed aortic OPN and Msx2 expression >50% and decreased cardiac valve calcification 80% (8.3 +/- 1.5% versus 1.4 +/- 0.5%; p < 0.001). Of the known circulating regulators of vascular calcification (OPN, osteoprotegerin, and leptin), PTH(1-34) regulated only serum OPN. We therefore studied actions of PTH(1-34) and OPN in vitro on cells induced to mineralize with Msx2. OPN (5-50 nM) reversed Msx2-induced mineralization. PTH(1-34) inhibited mineralization by 40% and down-regulated Msx2 in aortic myofibroblasts. PTH(1-34) inhibits vascular calcification and aortic osteogenic differentiation via direct actions and potentially via circulating OPN. PTH(1-34) exerts beneficial actions at early stages of macrovascular disease responses to diabetes and dyslipidemia.

Improvement of Barley yellow dwarf virus-PAV detection in single aphids using a fluorescent real time RT-PCR

One of the major factors determining the incidence of Barley yellow dwarf virus (BYDV) on autumn-sown cereals is the viruliferous state of immigrant winged aphids. This variable is assessed routinely using the enzyme-linked immunosorbant assay (ELISA). However, the threshold for virus detection by ELISA can lead to false negative results for aphids carrying less than 10 6 particles. Although molecular detection techniques enabling the detection of lower virus quantities in samples are available, the relatively laborious sample preparation and data analysis have restricted their use in routine applications. A gel-free real-time one-step reverse transcription polymerase chain reaction (RT-PCR) protocol is described for specific detection and quantitation of BYDV-PAV, the most widespread BYDV species in Western Europe. This new assay, based on TaqMan® technology, detects and quantifies from 10 2 to 10 8 BYDV-PAV RNA copies. This test is 10 and 10 3 times more sensitive than the standard RT-PCR and ELISA assays published previously for BYDV-PAV detection and significantly improves virus detection in single aphids. Extraction of nucleic acids from aphids using either phenol/chloroform or chelatin resin-based protocols allow the use of pooled samples or of a small part (up to 1/1600th) of a single aphid extract for efficient BYDV-PAV detection.

Gene expression profiling of cell lines derived from T-cell malignancies

The expression profiles of eight cell lines derived from T-cell malignancies were compared to CD4-positive T-cells using cDNA microarray technology. Unsupervised hierarchical clustering of 4364 genes demonstrated substantial heterogeneity resulting in four distinct groups. While no genes were found to be uniformly up- or down-regulated across all cell lines, we observed 111 over-expressed genes (greater than two-fold) and 1118 down-regulated genes (greater than two-fold) in the lymphomas as a group when compared to CD4-positive T-cells. These included genes involved in cytokine signaling, cell adhesion, cytoskeletal elements, nuclear transcription factors, and known oncogenes and tumor suppressor genes. Quantitative fluorescent reverse transcription-polymerase chain reaction analysis demonstrated 70% concordance with the microarray results. While freshly isolated malignant cells may differ in their individual expression patterns relative to established cell lines from the same diagnoses, we feel that the variety of different lymphocytic cell lines that we examined provides a representative picture of the molecular pathogenesis of T-cell malignancies.

Real Time RT-PCR Shows Correlation between Retinoid-Induced Apoptosis and NGF-R mRNA Levels

Neurotrophins and retinoic acid have a critical role in the differentiation and the survival of neurons. All-trans-, 9-cis-retinoic acid (10−6 M) or NGF (50–100 ng/ml) induced morphologic differentiation and inhibited cell growth in SH-SY5Y neuroblastoma cells after 7 days of culture. Continuous treatment of undifferentiated cells with all-trans- or 9-cis-retinoic (10−6 M) did not induce apoptosis, whereas NGF-differentiated cells showed dramatic apoptosis after 2 to 4 days of retinoic acid treatment as evidenced by TUNEL reaction and flow cytometry analysis following propidium iodide staining. Addition of Ro41-5253 blocked all-trans-retinoic-induced apoptosis, suggesting that the apoptotic signaling pathway was mediated by RARs. The effects of all-trans- or 9-cis-retinoic acid on the expression of NGF receptors was evaluated using real-time fluorescence reverse transcription-PCR. A slight transient increase in the expression of p75NGFR mRNA was observed by 2 to 4 h after retinoid treatment of undifferentiated cells, whereas a larger increase in the expression of both TrkA and p75NGFR mRNA up to threefold the basal level, was observed by 2 to 6 h after retinoid treatment of NGF-differentiated cells. Our results suggest that NGF-differentiated cells may be more susceptible to retinoid-induced apoptosis that undifferentiated cells.