Screening suitable reference genes is the premise of quantitative Real-time PCR(qRT-PCR)for gene expression analysis. To provide stable reference genes for expression analysis of genes in Aconitum vilmorinianum, this study selected 19 candidate re-ference genes(ACT1, ACT2, ACT3, aTUB1, aTUB2, bTUB, 18S rRNA, UBQ, eIF2, eIF3, eIF4, eIF5, CYP, GAPDH1, GAPDH2, PP2A1, PP2A2, ACP, and EF1α) based on the transcriptome data of A. vilmorinianum. qRT-PCR was conducted to profile the expression of these genes in the root, stem, leaf, and flower of A. vilmorinianum. The Ct values showed that 18S rRNA with high expression level and GAPDH2 with large expression difference among organs were not suitable as the reference genes. NormFinder and geNorm showed similar results of the expression stability of the other candidate reference genes and demonstrated PP2A1, EF1α, and CYP as the highly stable ones. However, BestKeeper suggested EF1α, ACT3, and PP2A1 as the top stable genes. In view of the different results from different softwares, the geometric mean method was employed to analyze the expression stability of the candidate re-ference genes, the results of which indicated that PP2A1, EF1α, and ACT3 were the most stable. Based on the comprehensive analysis results of geNorm, NormFinder, BestKeeper, and geometric mean method, PP2A1 and EF1α presented the most stable expression in different organs of A. vilmorinianum. PP2A1 and EF1α were the superior reference genes for gene expression profiling in different organs of A. vilmorinianum.
Aconitum ; Gene Expression Profiling ; Genes, Plant/genetics* ; Real-Time Polymerase Chain Reaction ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo prepare the armored RNA containing M gene of influenza H3N2.

METHODSThe vector pAR-1 was constructed from expression vector pET30b in which the bacteriophage MS2 DNA fragment, containing the genes for maturase and coat protein and the pac site, was inserted. The M gene fragment of influenza A was inserted into the HindIII site downstream of the pac site on the pAR-1, which formed a new recombinant plasmid pAR-2. After the prokaryotic expression was carried out, armored RNA AR-2 containing M gene was obtained. AR-2 was purified, and then was quantified by real time RT-PCR. Moreover, the stability of AR-2 was checked.

RESULTSAR-2 was expressed successfully. AR-2 remained stable under various storage environments. Approximately 8.9 x 10(11) copies of AR-2 particles can be purified from one milliliter of culture.

CONCLUSIONIt showed that AR-2 was stable and RNase-resistant, which, as a virus surrogate, would be used as RT-PCR standards, controls and training or proficiency samples.

Influenza A Virus, H3N2 Subtype ; genetics ; Plasmids ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; standards ; Viral Matrix Proteins ; genetics

To rapidly identify the cross-contamination problems caused by the positive control in the process of mumps virus nucleic acid detection, a new mumps virus RNA positive control was developed in this study. Using the same primers and reaction conditions, the cross-contamination problems caused by the positive control could be readily identified by comparing the fragments lengths of the PCR products between the positive control and the samples. This new RNA positive control of mumps virus can be widely used in the diagnosis and genotyping of mumps virus as a better laboratory quality control.
Cell Line ; DNA Primers ; genetics ; standards ; Genotype ; Humans ; Mumps ; diagnosis ; virology ; Mumps virus ; genetics ; isolation & purification ; RNA, Viral ; genetics ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; standards

Viral load testing of human immunodeficiency virus (HIV) is an essential tool for initiating and monitoring the antiretroviral therapy for HIV patients. To this end, several methods including polymerase chain reaction (PCR), branched DNA (bDNA), nucleic acid sequence based amplification assay (NASBA) and internally controlled virion PCR (ICV PCR) have become available. Of these methods, the standard reverse transcription-PCR (RT-PCR) assay has been widely used in Korea. However, no comparison study has been performed among the various detection methods currently used in Korean patients. We evaluated the correlation and agreement between the PCR and the branched DNA (bDNA) assay for measurement of HIV RNA in Korean patients. Eighty randomly selected samples from HIV-1-seropositive patients visiting Yonsei Medical Center Severance Hospital were studied. We found that these assays show good agreement, have a reliable correlation (r = 0.92, mean difference in log10 copies/ mL +/- 2 standard deviation = 0.098 +/- 0.805) and produce values whose relationship is given by the following equation: log10v3 bDNA = -0.3405 + 1.0601 x log10RT-PCR. Thus, we conclude that these two methods may allow direct comparison of the results obtained from different assay systems.
Comparative Study ; DNA/chemistry* ; DNA/analysis* ; Genetic Techniques/standards* ; HIV-1/genetics* ; Human ; Korea ; RNA, Viral/blood* ; Reverse Transcriptase Polymerase Chain Reaction/standards*

OBJECTIVEDevelopment of pseudoviral competitive internal controls for RT-PCR laboratory detection of dengue virus.

METHODSThe internal controls target gene were obtained by insertion of a 180 bp non-related DNA fragment into RT-PCR detection target of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions.

RESULTSThe established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis.

CONCLUSIONThe pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus.

Cell Line ; DNA, Viral ; genetics ; Dengue ; diagnosis ; virology ; Dengue Virus ; genetics ; isolation & purification ; Humans ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; standards

OBJECTIVETo validate the conversion factor (CF) for the conversion of BCR-ABL (P210) transcript levels to the international scale in chronic myeloid leukemia (CML).

METHODSIn 2012, the international reference laboratory in Adelaide, Australia (IMVS) sent two batches of RNA samples, 30 samples per batch, to Peking University People's Hospital (PKUPH). By comparing BCRABL (P210) transcript levels reported by the two laboratories, CF of PKUPH was calculated and validated by IMVS. In 2013, PKUPH prepared the exchange samples for validation of CF of 9 hospitals who have calculated CFs before. The fresh BCR-ABL (P210) (+) cells were serially diluted by BCR-ABL (P210) (-) cells to prepare 22 kinds of samples with different BCR-ABL transcript levels, each kind had 10 parallel samples. Trizol reagent was added in each tube. Ten hospitals tested BCR-ABL transcript levels of one set of 22 samples. Agreement between BCR-ABL transcript levels of each laboratory and PKUPH was assessed by the Bland-Altman method.

RESULTSPKUPH successfully validated its CF with bias 1.1 fold and 95% limits of agreement between -4.7 and 4.9 fold. Of 9 hospitals whose validation performed by sample exchanges with PKUPH, 6 hospitals successfully validated their CF with bias ≤±1.4 fold and 95% limits of agreement within ±6 fold.

CONCLUSIONValidation of CF examined the stability of the detection of BCR-ABL (P210) transcript levels, which was necessary for the valid conversion of BCR-ABL (P210) transcript levels to the international scale in CML.

Fusion Proteins, bcr-abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; standards ; Transcription, Genetic

As a traditional Chinese medicine, Senecio scandens is rich in important compounds such as flavonoid and sesquiterpenoid. Based on the transcriptome data of S. scandens, 15 candidate reference genes were selected including ABCT, ACT1, ACT2, ACT3, ACBP, ARF, ATPS, EF-H, EF-1α, ETIF, GAPDH, GTPB, MPS, UCE and 60S. Firstly, 9 candidate genes with relatively stable expressions such as ACT1, ACBP, ARF, ATPS, EF-1α, GAPDH, MPS, UCE and 60S were screened from different tissues of S. scandens by RT-PCR. Then, qRT-PCR was used to quantitatively analyze gene expression of these nine candidates in S. scandens with or without stress treatments. Further analysis of these gene expression data by geNorm and NormFinder showed that ACT1 exhibited the stablest expression in all samples and could serve as a reference gene for future study of S. scandens, and provide an endogenous control for gene expression analysis.
Gene Expression Profiling ; Genes, Plant ; Medicine, Chinese Traditional ; Plants, Medicinal ; genetics ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction ; Senecio ; genetics ; Transcriptome

Quantitative real-time RT-PCR (RT-qPCR) is being widely used in microRNA expression research. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in microRNA RT-qPCR studies. The aim of this study was to identify the most stable reference gene(s) for quantification of microRNA expression analysis in uterine cervical tissues. A microarray was performed on 6 pairs of uterine cervical tissues to identify the candidate reference genes. The stability of candidate reference genes was assessed by RT-qPCR in 23 pairs of uterine cervical tissues. The identified most stable reference genes were further validated in other cohort of 108 clinical uterine cervical samples: (HR-HPV- normal, n = 21; HR-HPV+ normal, n = 19; cervical intraepithelial neoplasia [CIN], n = 47; cancer, n = 21), and the effects of normalizers on the relative quantity of target miR-424 were assessed. In the array experiment, miR-26a, miR-23a, miR-200c, let-7a, and miR-1979 were identified as candidate reference genes for subsequent validation. MiR-23a was identified as the most reliable reference gene followed by miR-191. The use of miR-23a and miR-191 to normalize expression data enabled detection of a significant deregulation of miR-424 between normal, CIN and cancer tissue. Our results suggested that miR-23a and miR-191 are the optimal reference microRNAs that can be used for normalization in profiling studies of cervical tissues; miR-23a is a novel microRNA normalizer.
Cervical Intraepithelial Neoplasia/diagnosis/genetics/*metabolism/pathology ; Cervix Uteri/*metabolism/pathology ; Early Detection of Cancer ; Female ; Gene Expression Profiling/*standards ; Humans ; MicroRNAs/genetics/*metabolism/standards ; Microarray Analysis ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction ; Uterine Cervical Neoplasms/diagnosis/genetics/*metabolism/pathology

OBJECTIVE: To investigate the clinical outcome of patients with moderate type of corona virus disease 2019 (COVID-19) after discharge by retesting viral nucleic acid. METHODS: Seven patients with moderate COVID-19 met the discharge criteria enacted by National Health Commission were quarantine in hospital for 7 days, then continuously quarantined at home for 4 weeks after discharged. During the three weeks of quarantined period, the symptoms and signs were documented; and sputum or nasal swab and feces samples were collected to test SARS-COV-2 nucleic acid by RT-PCR method. RESULTS: There were no symptoms and signs during the quarantine period in all 7 patients. However, respiratory swabs from 3 patients were confirmed positive of SARS-COV-2 nucleic acid at 5 to 7 days after they met the discharge criteria. CONCLUSIONS The study indicates that there is a relatively high incidence of positive viral nucleic acid in patients met the discharge criteria, and it is suggested that patients met the current discharge criteria should be quarantined in hospital for another 7 days and the follow-up viral testing is necessary.
Asymptomatic Diseases ; Betacoronavirus ; genetics ; China ; Clinical Laboratory Techniques ; Coronavirus Infections ; diagnosis ; virology ; Follow-Up Studies ; Humans ; Pandemics ; Patient Discharge ; standards ; Pneumonia, Viral ; diagnosis ; virology ; Quarantine ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

To gain molecular understanding of carcinogenesis of breast cancer, gene expression profiles were analyzed using cDNA microarray representing 4,600 cDNAs in 10 breast cancer samples and the adjacent noncancerous breast tissues from the same patients. The alterations in gene expression levels were confirmed by reversetranscription PCR in four randomly selected genes. Genes that were differently expressed in cancer and noncancerous tissues were identified. 106 (of which 55 were known) and 49 (of which 28 were known) genes were up- or down-regulated, respectively, in greater than 60% of the breast cancer samples. In cancer tissues, genes related to cell cycle, transcription, metabolism, cell structure/motility and signal transduction were mostly up-regulated. Furthermore, three cancer tissues showing immunohistochemically aberrant accumulation of beta-catenin in the nucleus and/or cytoplasm revealed down-regulation of Siah and Axin genes and up-regulation of Wnt and c-myc genes. These findings were highly consistent with Wnt signaling pathway associated with beta-catenin regulation previously suggested by others. Our studies, therefore, provide not only a molecular basis to understand biological processes of breast cancer but also useful resources to define the mechanism of beta-catenin expression in tumorigenesis of breast cancer.
Adult ; Breast Neoplasms/*genetics/*metabolism/pathology ; Cytoskeletal Proteins/*metabolism ; Female ; *Gene Expression Profiling/standards ; Gene Expression Regulation, Neoplastic ; Human ; Immunohistochemistry ; Middle Aged ; *Oligonucleotide Array Sequence Analysis/standards ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Support, Non-U.S. Gov't ; Trans-Activators/*metabolism