Paeonia veitchii and P. lactiflora are both original plants of the famous Chinese medicinal drug Paeoniae Radix Rubra in the Chinese Pharmacopoeia. They have important medicinal value and great potential in the flower market. The selection of stable and reliable reference genes is a necessary prerequisite for molecular research on P. veitchii. In this study, two reference genes, Actin and GAPDH, were selected as candidate genes from the transcriptome data of P. veitchii. The expression levels of the two candidate genes in different tissues(phloem, xylem, stem, leaf, petiole, and ovary) and different growth stages(bud stage, flowering stage, and dormant stage) of P. veitchii were detected using real-time fluorescence quantitative technology(qRT-PCR). Then, the stability of the expression of the two reference genes was comprehensively analyzed using geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that the expression patterns of Actin and GAPDH were stable in different tissues and growth stages of P. veitchii. Furthermore, the expression levels of eight genes(Pv-TPS01, Pv-TPS02, Pv-CYP01, Pv-CYP02, Pv-CYP03, Pv-BAHD01, Pv-UGT01, and Pv-UGT02) in different tissues were further detected based on the transcriptome data of P. veitchii. The results showed that when Actin and GAPDH were used as reference genes, the expression trends of the eight genes in different tissues of P. veitchii were consistent, validating the reliability of Actin and GAPDH as reference genes for P. veitchii. In conclusion, this study finds that Actin and GAPDH can be used as reference genes for studying gene expression levels in different tissues and growth stages of P. veitchii.
Real-Time Polymerase Chain Reaction/methods* ; Paeonia/genetics* ; Actins/genetics* ; Reproducibility of Results ; Transcriptome ; Glyceraldehyde-3-Phosphate Dehydrogenases/genetics* ; Reference Standards ; Gene Expression Profiling/methods*

BACKGROUND: A nucleic acid amplification test was adopted to detect transfusion-transmitted infectious agents. In the case of HTLV, however, there was no internal control (IC) because the laboratory developed polymerase chain reaction (laboratory-developed PCR) was used. In this study, noncompetitive IC was constructed for the laboratory-developed PCR of HTLV and the effectiveness was compared with the competitive test that was constructed in a previous study. METHODS: As a competitive IC, plasmid DNA, including the primer recognition sequence for the amplification of the HTLV pX region, was constructed. As a noncompetitive IC, an additional primer was constructed for the amplification of the housekeeping gene, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. The performance of the competitive and noncompetitive IC was verified and compared using 10 HTLV positive samples and 10 negative samples. In addition, the detection limits in the assay adopting competitive IC and noncompetitive IC were compared. RESULTS: In the case of competitive IC applications, all 10 positive samples were positive and all 10 negative samples were negative. In the case of noncompetitive IC applications, however, one positive sample was not detected. The detection limit of the assay using competitive IC was 100 pg and that of the assay using noncompetitive IC was 1 ng. CONCLUSION: Although the manufacturing processes is not required using noncompetitive IC, the adoption of competitive IC is more effective to ensure the assay results because the ability of detection of the assay adopting competitive IC was better than that using noncompetitive IC.
DNA ; Genes, Essential ; Glyceraldehyde 3-Phosphate ; Limit of Detection ; Nucleic Acid Amplification Techniques ; Oxidoreductases ; Plasmids ; Polymerase Chain Reaction

OBJECTIVES.: Sodium salicylate (SS) is well known for its ototoxic properties that induce functional and morphological changes in the cochlea and brain. Ginkgo biloba extract (GBE) has been widely used for treatment of various neurodegenerative diseases; however, its effects on salicylate-induced ototoxicity remain unclear. Herein, we examined the effects of EGb 761 (EGb), a standard form of GBE, on the plasticity of the N-methyl-D-aspartate receptor subunit 2B (GluN2B) in the inferior colliculus (IC) following SS administration. METHODS.: Seven-week-old Sprague Dawley rats (n=24) were randomly allocated to control, SS, EGb, and EGb+SS groups. The SS group received a single intraperitoneal SS injection (350 mg/kg), the EGb group received EGb orally for 5 consecutive days (40 mg/kg), and the EGb+SS group received EGb for 5 consecutive days, followed by an SS injection. The auditory brainstem responses (ABRs) were assessed at baseline and 2 hours after SS administration. GluN2B expression was examined by Western blot and immunohistochemistry. RESULTS.: There were no significant differences in ABR threshold shifts among the groups. The expression of the GluN2B protein normalized by which of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was significantly lower in the EGb+SS group, as compared to the SS group (P=0.012). Weak and diffused GluN2B immunoreactivity was detected in the IC neural cells of the EGb+SS group, while those of the SS group exhibited strong and diffused GluN2B positivity. CONCLUSION.: EGb may play a role in regulating the GluN2B expression in the IC of salicylate-induced ototoxicity model.
Blotting, Western ; Brain ; Cochlea ; Evoked Potentials, Auditory, Brain Stem ; Ginkgo biloba ; Glyceraldehyde 3-Phosphate ; Immunohistochemistry ; Inferior Colliculi ; N-Methylaspartate ; Neurodegenerative Diseases ; Oxidoreductases ; Plastics ; Rats, Sprague-Dawley ; Sodium Salicylate

PURPOSE: The purpose of this study was to evaluate the effects of 1,25-dihydroxyvitamin D₃ on the proliferation, differentiation, and matrix mineralization of MC3T3-E1 osteoblast-like cells in vitro. METHODS: MC3T3-E1 osteoblastic cells and 1,25-dihydroxyvitamin D₃ were prepared. Cytotoxic effects and osteogenic differentiation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, alkaline phosphatase (ALP) activity assay, ALP staining, alizarin red S staining, and reverse transcription-polymerase chain reaction (RT-PCR) for osteogenic differentiation markers such as ALP, collagen type I (Col-I), osteocalcin (OCN), vitamin D receptor (VDR), and glyceraldehyde 3-phosphate dehydrogenase. RESULTS: The MTT assay showed that 1,25-dihydroxyvitamin D₃ did not inhibit cell growth and that the rate of cell proliferation was higher than in the positive control group at all concentrations. ALP activity was also higher than in the positive control group at low concentrations of 1,25-dihydroxyvitamin D₃ (10−10, 10−12, and 10−14 M). RT-PCR showed that the gene expression levels of ALP, Col-I, OCN, and vitamin D receptor (VDR) were higher at a low concentration of 1,25-dihydroxyvitamin D₃ (10−12 M). Alizarin red S staining after treatment with 1,25-dihydroxyvitamin D₃ (10−12 M) showed no significant differences in the overall degree of calcification. In contrast to the positive control group, formation of bone nodules was induced in the early stages of cell differentiation. CONCLUSIONS: We suggest that 1,25-dihydroxyvitamin D₃ positively affects cell differentiation and matrix mineralization. Therefore, it may function as a stimulating factor in osteoblastic bone formation and can be used as an additive in bone regeneration treatment.
Alkaline Phosphatase ; Antigens, Differentiation ; Bone Regeneration ; Calcitriol ; Cell Differentiation ; Cell Proliferation ; Collagen Type I ; Gene Expression ; Glyceraldehyde 3-Phosphate ; In Vitro Techniques ; Miners ; Osteoblasts ; Osteocalcin ; Osteogenesis ; Oxidoreductases ; Receptors, Calcitriol

GAPDH(glyceraldehyde-3-phosphate dehydrogenase) gene is a key enzyme gene in carbohydrate metabolism and always used as reference gene. To clarify and complete the biosynthetic pathway of polysaccharide, the GAPDH gene in Codonopsis pilosula, named CpGAPDH, was cloned according to the transcriptome of pilosula, using the GAPDH gene in potato as query. The CpGAPDH contained a 1 014 bp open reading frame(ORF) and encoded a protein with 337 amino acids. Bioinformatic analysis clearly suggested that CpGAPDH shared high similarity with GAPDH among other plants, and had the closest relatives to potato and danshen. The predicted protein did not have signal peptide, which indicated that it might be located in the cytoplasm. According to the existing of several phosphorylation sites and the conserved domains analysis, we predicted that it belonged to Gp_dh_N superfamily. Prokaryotic expression showed that the recombinant expressed a 44.3 kDa protein, which was corresponding to the theoretical relative molecular mass. However, the relative transcript level of the CpGAPDH did not have significant differences in different tissues and roots at different developmental stages of pilosula. Moreover, the stability of the CpGAPDH was analyzed by BestKeeper, geNorm, and NormFinder and RefFinder software, which showed that the CpGAPDH was more stable and could be used as a new reference gene. All these lay a foundation for the expression analysis of the gene relative to the polysaccharide synthesis.
Codonopsis ; enzymology ; genetics ; Glyceraldehyde-3-Phosphate Dehydrogenases ; genetics ; Plant Proteins ; genetics ; Polysaccharides ; biosynthesis ; Transcriptome

A seed-borne fungus, Curvularia sp. EML-KWD01, was isolated from an indigenous wheat seed by standard blotter method. This fungus was characterized based on the morphological characteristics and molecular phylogenetic analysis. Phylogenetic status of the fungus was determined using sequences of three loci: rDNA internal transcribed spacer, large ribosomal subunit, and glyceraldehyde 3-phosphate dehydrogenase gene. Multi loci sequencing analysis revealed that this fungus was Curvularia spicifera within Curvularia group 2 of family Pleosporaceae.
Classification* ; DNA, Ribosomal ; Fungi ; Glyceraldehyde 3-Phosphate ; Humans ; Oxidoreductases ; Ribosome Subunits, Large ; Triticum

The basidiomycete Laetiporus sulphureus var. miniatus belongs to the Aphyllophorales, Polyporaceae, and grows on the needleleaf tree. The fruiting bodies of Laetiporus species are known to produce N-methylated tyramine derivatives, polysaccharides, and various lanostane triterpenoids. As part of our ongoing effort to discover biologically active compounds from wood-rotting fungi, an anti-inflammatory triterpene, LSM-H7, has been isolated from the fruiting body of L. sulphureus var. miniatus and identified as acetyl eburicoic acid. LSM-H7 dose-dependently inhibited the NO production in RAW 264.7 cells without any cytotoxicity at the tested concentrations. Furthermore it suppressed the production of proinflammatory cytokines, mainly inducible nitric oxide synthase, cyclooxygenase-2, interleukin (IL)-1beta, IL-6 and tumor necrosis factor alpha, when compared with glyceraldehyde 3-phosphate dehydrogenase. These data suggest that LSM-H7 is a crucial component for the anti-inflammatory activity of L. sulphureus var. miniatus.
Basidiomycota ; Cyclooxygenase 2 ; Cytokines ; Fruit ; Fungi ; Glyceraldehyde 3-Phosphate ; Inflammation* ; Interleukin-6 ; Interleukins ; Macrophages* ; Nitric Oxide ; Nitric Oxide Synthase Type II ; Oxidoreductases ; Polyporaceae ; Polyporales ; Polysaccharides ; Trees ; Tumor Necrosis Factor-alpha ; Tyramine

BACKGROUND: Because of recent advances in the molecular diagnosis of cancer patients, tissue quality has become more important in daily practice. METHODS: To evaluate the effects of fixative, duration of fixation, decalcification, and storage periods on nucleic acid integrity, DNA and RNA were extracted from gastrointestinal cancer tissue. The yield and purity were analyzed, and polymerase chain reaction (PCR) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 60 bp), beta-actin (148 bp), and human growth hormone (hGH; 434 bp) and real-time reverse transcription-PCR for beta-actin (97 bp) were performed. RESULTS: All formalin-fixed paraffin-embedded (FFPE) and methacarn-fixed paraffin-embedded (MFPE) samples tested positive for GAPDH and beta-actin by PCR. hGH was successfully detected in all MFPE samples, but in only 46.7% of the FFPE samples. Prolonged formalin fixation resulted in fewer GAPDH and beta-actin PCR products, and amplification of hGH was not successful. The PCR and reverse transcription-PCR results were significantly affected by the duration of decalcification. The yield, purity, and integrity of mRNA progressively decreased with increased storage periods of paraffin blocks. CONCLUSIONS: Fixation and storage should therefore be standardized in order to improve the quality of molecular pathologic diagnosis.
Actins ; Diagnosis ; DNA ; Formaldehyde ; Gastrointestinal Neoplasms ; Glyceraldehyde 3-Phosphate ; Human Growth Hormone ; Humans* ; Oxidoreductases ; Paraffin ; Polymerase Chain Reaction ; RNA ; RNA, Messenger

OBJECTIVE: To observe the changes of relative expression of myocardial various RNAs in rats died of different causes and their relationship with PMI. METHODS: The rat models were established in which the rats were sacrificed by broken neck, asphyxia, and hemorrhagic shock. Total RNAs were extracted from myocardium. The quantitative real time PCR was used to calculate threshold cycle values of RNAs including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, inducible nitric oxide synthase (iNOS), hypoxia-inducible factor-1 (HIF-1), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and U6 small nuclear RNA (U6 snRNA) and to study the changes of the relative expressions of various indexes with PMI. RESULTS: U6 snRNA with stable expression level could be used as appropriate internal control. In the early PMI, the relative expression of GAPDH, HIF-1, iNOS, TNF-alpha, and IL-6 more characteristically increased in groups of asphyxia and hemorrhagic shock than in group of broken neck, but the quantity of beta-actin decreased in all groups. In the late PMI, all the relative expressions significantly declined in correlation with the degradation of RNA. CONCLUSION The characteristic changes of each RNA expression can be used as references to estimate PMI in deaths by different causes.
Actins ; Animals ; Cause of Death ; Cytokines/metabolism* ; Disease Models, Animal ; Enzymes/metabolism* ; Glyceraldehyde-3-Phosphate Dehydrogenases ; Myocardium/metabolism* ; Nitric Oxide Synthase Type II ; RNA/metabolism* ; RNA, Small Nuclear ; Rats ; Shock, Hemorrhagic ; Tumor Necrosis Factor-alpha

OBJECTIVE: To explore the correlation between postmortem interval (PMI) and five RNA markers of rat's skin--β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA(18S rRNA), 5S ribosomal RNA (5S rRNA), and microRNA-203 (miR-203), at different temperatures. METHODS: Eighteen SD rats were randomly divided into three environmental temperature groups: 4 °C, 15 °C and 35 °C, respectively. Skin samples were taken at 11 time points from 0 h to 120 h post-mortem. The total RNA was extracted from the skin samples and the five RNA levels were detected by real-time fluorescent quantitative PCR. Proper internal reference was selected by geNorm software. Regression analysis of the RNA markers was conducted by GraphPad software. RESULTS: 5S rRNA and miR-203 were most suitable internal references. A good linear relationship between PMI and RNA levels (β-actin and GAPDH) was observed in two groups (4 °C and 15 °C), whereas the S type curve relationship between the expression levels of the two markers (β-actin and GAPDH) and PMI was observed in the 35 °C group. The partial linear relationship between 18S rRNA and PMI was observed in the groups (15 °C and 35 °C). CONCLUSION Skin could be a suitable material for extracting RNA. The RNA expression levels of β-actin and GAPDH correlate well with PMI, and these RNA markers of skin tissue could be additional indice for the estimation of PMI.
Actins ; Animals ; Autopsy ; Glyceraldehyde-3-Phosphate Dehydrogenases/genetics* ; Postmortem Changes ; RNA ; RNA Stability ; RNA, Ribosomal, 18S ; Rats ; Real-Time Polymerase Chain Reaction ; Regression Analysis ; Skin ; Temperature