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Objective To establish the method of the SYBR Green Ⅰ real‐time fluorescent quantitative PCR for detecting the se‐rum miR‐203 expression level ,and to detect the serum miR‐203 expression levels in the patients with cervical cancer ,cervical benign diseases and healthy controls .Methods The miR‐203 ,U6 stem loop RT primers and the PCR amplification primers were designed for conducting fluorescence quantitative PCR ,with U6 as the internal relative quantification ,the serum miR‐203 levels were com‐pared among different cervical diseases .Results The established method could specifically detect the amplification signal of serum miR‐203 ,the melting curve was single and PCR products were specific .The serum miR‐203 level in the patients with cervical cancer was significantly higher than that in the patients with benign cervical diseases such as hysteromyoma and cervicitis ,the difference was statistically significant (P<0 .05) .Conclusion The SYBR Green Ⅰ real‐time fluorescent quantitative PCR is a quick ,simple detection method with high sensitivity and good specificity ,which may have a better application prospect in cervical cancer auxiliary diagnosis .
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A novel fluorescence homogeneous biosensing strategy was developed for simple, rapid and sensitive detection of melamine in milk by using the polythymine oligonucleotide T24 and SYBR Green Ⅰ. In the absence of melamine, the fluorescence of SYBR Green Ⅰ was weak. The interactions between the single strand oligonucleotide T24 and SYBR Green Ⅰ were weak. In contrast, the presence of melamine drove the formation of T-melamine-T folded structure and enabled the SYBR Green Ⅰ to intercalate into double-strand DNA, resulting in the enhancement of fluorescence intensity. The results revealed that the method allowed a sensitive, simple, and rapid assay of melamine with a linear response range from 0. 1 μmol/L to 10 μmol/L and a detection limit of 35 nmol/L.
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Objective To investigate the effects of Lipo-PGE1 on the expression of T-bet and Gata-3,and its potential mechanisms causing the shift of T cells from Th1 to Th2 on Acute lung injury(ALI)induced by Lipopolysaccharide(LPS)in mice.Methods Sixty male BALB-C mice were randomly divided into three groups(n =20 in each group):(1)control group,mice were treated with intravenous injection of NS in dose of 10 ml/kg,(2)LPS group,mice were exposed to LPS with dosage of 5 mg/kg(0.5 g/ml diluted in saline),and(3)LPS + PGE1 group,mice were treated with Lipo-PGE1 in dose of 15μg/kg.Sixhours after injection,the lungs were removed for observing the histopathological changes and determination of wet/dry lung weight(W/D)ratio.The levels of Th1 and Th2 were determined by flow cytometry,and the expressions of T-bet and Gata-3 mRNA were detected by using RT-PCR.One-way ANOVA was used for comparing differences between groups,and all data were presented in((x)± s).Results The histological changes of lung injury were lessened by PGEC ompared with the W/D ratio(5.74 ± 0.31)in LPS group,the one(4.92 ±0.27)in LPS +PGE1 group was lower significantly(P ＜0.01).The levels of Th1 and Th2 and their ratio Were higher in LPS +PGE1 group[(20.31 ±2.20)％,(10.50±0.80)％,(1.93±0.05)]than in LPS group[(16.65 ±1.70)％,(9.40 ±1.25)％,(1.73 ±0.03)](P＜0.01).Compared with control group,the expressions ofT-bet mRNA(1.183 ±0.495),and Gata-3 mRNA(0.693±0.285),and their ratio(1.713 ± 0.131)were lower(P ＜0.01); compared with LPS group,PGE1 significantly increased the expressions of T-bet mRNA(1.827 ± 0.705)and the ratio of T-bet/Gata-3 (2.502 ±0.352)(P ＜0.01),while didn(t)increased the expressions of Gata-3 mRNA(0.7191 ±0.186)significantly(P ＞ 0.05).Conclusions Lipo-PGE1 may up-regulate transcription factor T-bet which participates in the Th1 differentiation ratio,and then improve the inflammatorv svmntom.
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Objective To establish a method to detect the glutathione stransferase(GST) M1 gene polymorphisms rapidly and to apply preliminary for detection.Methods GSTM1 gene and internal control gene CYP1A1 were amplified by multi-PCR with appropriate fluorescent dye SYBR green I in the reaction system,The changes of fluorescence values were recorded from 65 ℃ to 95 ℃ by a rate of(0.1 ℃/s) when PCR was just completed.Results There were two peaks in the melting curve of GSTM1~+ genotype,but only a single peak occurred for the GSTM1-genotype.The temperatures of two peaks corresponded to the expected Tm.agarose gel electrophoresis analysis demonstrated that these peaks correspond to the bands of the predicted molecular size.The detection can be completed within an hour after DNA was extracted.Conclusion Melting curve analysis combined with SYBR green I is a easy,rapid,accurate methodfor detection of GSTM1 gene polymorphisms.
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Objective To analyze the variation of intestinal microflora in patients with colorectal cancer by SYBR GreenⅠreal-time fluorescence quantitative PCR and reveal the role and significance of intestinal microflora in the colorectal cancer-associated molecular pathogenesis.Methods A set of 16S rRNA gene group of species-specific primers for Bifidobacterium spp.,Lactobacillus group,Escherichia coli,and ddl gene-targeted species-specific primers for Enterococcus faecalis and feces Enterococcus were designed.Patients with colorectal cancer(colorectal cancer group,n=30) and healthy volunteers(normal control group,n=30) were included and whose feces were collected to extract bacterial genome DNA.SYBR GreenⅠ real-time fluorescence quantitative PCR was used to analyze the five mentioned bacterial amounts.Results Level of Bifidobacterium spp.(4.52?0.49) and Lactobacillus group(5.46?0.12) in colorectal cancer group were significantly lower than those(9.25?0.83 and 7.45?0.37) of normal control group(P
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Objective To establish a SYBR Green Ⅰ quantitative real-time PCR method for detecting the expression of APRIL gene(a proliferation-inducing ligand,APRIL) in peripheral blood of patients with auto-immune diseases,e.g.,systemic lupus erythematosus(SLE) and rheumatoid arthritis(RA),and investigate the relationship of APRIL mRNA expression with pathogenesis and prognosis of auto-immune diseases.Methods Plasmid PGEM-T easy-APRIL was cloned as the standard template.SYBR Green Ⅰ quantitative real-time PCR was set up to examine the expression of APRIL mRNA in peripheral blood of 58 patients with auto-immune diseases and 20 healthy controls using Line Gene FQD-33A Detection System.Results The obtained data were normalized by dividing the copy number of target cDNA by those of GAPDH(glyceraldehycle-3-phosphate dehydrogenase,GAPDH).APRIL expression levels ranges from 3.95 to 192 and mean value was 29.68?4.5.APRIL expression of twenty healthy controls showed range from 3.1 to 18.7 and mean value of 10.56?2.0.APRIL expression levels in patients with auto-immune diseases were higher than those in healthy controls.In auto-immune diseases group APRIL expression levels of untreated patients were higher than those of the other patients,and a statistical significance was found.Conclusions APRIL mRNA was successfully detected by SYBR Green Ⅰ quantitative real-time PCR and the method was accurate and reliable.The expression of APRIL mRNA of auto-immune disease patients was higher than those of healthy controls,and the expression of untreated patients was the highest.This method may be used for further study on the high-level expression of APRIL mRNA in mechanism of auto-immune diseases as well as the development and prognosis of diseases.