3.Study on the amplifying and typing of low copy number DNA
Chinese Journal of Forensic Medicine 2009;24(6):383-385,403
Objective To study the influence on STR typing for low copy number(LCN)DNA using different methods of amplifcation and detection.Methods Control DNA 9947A was diluted and then amplifled with Identifiler~(TM) or DNATyper15~(TM).The heat cycles were set to 28 or 28 add 6.Each template wag amplified three times in parallel,and then the amplified products or the product mixture of three amplifictions were deteced with 310 or 3130 Genetic Analyzer.Results The success rate of STR typing with the method 28+6 cycles was higher than that of 28 cycles.There are no correlation between the allele imbalance or allele dropout and STR locus.With the reduction in the amount of template DNA,allele imbalance and dropout gradually increased,and the allele dropout was more common than allele imbalance when the amount of template DNA Wag very small.When the product mixture of three amplifictions were deteced.the occurrence of allele imbalance and dropout reduce obviously.Conclusion The success rate of STR typing of LCN DNA can be obviously increased by detecting the product mixture of three amplifictions in parallel combined with the 28+6 heat cycle condition.
4.Preparation of internal quality control substance of real-time PCR to analyze UU-DNA and the design quality control programs
Yanmei GE ; Di ZHANG ; Suyi FAN ; Hang YUAN ; Yuan MAO
International Journal of Laboratory Medicine 2016;37(8):1070-1071,1074
Objective Developing an internal quality control substance of Ureaplasma urealyticum(UU)‐DNA for real‐time PCR to establish an internal quality control system and preliminary evaluation its clinical value .Methods Internal quality control sub‐stance was prepared by mixing samples which Ct value were 24-25(positive sample) and 32 -33(weak positive sample) ,respec‐tively .At the same time ,selecting samples that test results were negative as negative control .The target value ,standard deviation (s) and coefficient of variation(CV) of internal quality control substance were defined by“instant method”for the first 20 runs and Levey‐Jennings quality control(QC) chart after the first 20 runs .Using the“Westdard” multi‐rule quality control methods to ana‐lyze the detection results .Exporting OPSPecs chart by quality control rules in Unity Real Time (URT ) system and setting up new quality control rules according with OPSPecs chart .Results 131 times of the detection of quality control substance were performed totally .The first 20 runs were defined by“instant method”and later 111 runs were defined by Levey‐Jennings QC chart ,the results were stable of quality control substance and reasonable quality control rules .Conclusion Preparing of internal quality control sub‐stance of UU‐DNA used in real‐time PCR might be easy and stable .So ,the internal quality control substance of UU‐DNA could be worthy for practical application in this PCR laboratory .Design internal quality control rules based OPSPecs chart in molecular de‐tection is very simple and practical .
5.Expression of miRNA-1247-3P in acute respiratory distress syndrome cell model and its function
Dongliang CHENG ; Yuan LIANG ; Yanchen CHEN ; Di QING ; Changsong SHI
The Journal of Practical Medicine 2015;(17):2802-2804
Objective To detect the expression pattern of microRNA in A549 cells treated by lipopolysaccharide, study the expression of miRNA-1247-3P in A549 cells under LPS treatment and explore the possible mechanism of miRNA-1247-3P in A549 cells under LPS treatment. Methods A549 cells were divided into experimental and control groups. Immunocytochemical method and RT-PCR were used to detect the changes of SP-A and SP-C. The expression of miRNAs were detected using miRNAs array in different groups. The key miR-1247-3P was collected to detect the changes of miR-1247-3P in all groups using quantitative real-time polymerase chain reaction. Results Compared with control group, the expressions of SP-A and SP-C were significantly decreased in the experimental groups (P < 0.05). MiRNA array showed that 31 miRNAs were up-regulated and 3 miRNAs were down-regulated. Compared with control group, the expression of miR-1247-3P was significantly increased in the experimental groups (P < 0.05). Conclusion The increased expression of miR-1247-3P may play an important role in the pathogenesis of ARDS.
6.The Effects of UrsoIic Acid on CispIatin-Induced Expression of Transient Receptor PotentiaI VaniIIoid 1 in Mouse CochIea
Yang DI ; Li YU ; Yuan TIAN ; Yuhan LIN ; Aimei WANG
Journal of Audiology and Speech Pathology 2015;(1):57-60
Objective To investigate the effects of ursolic acid (UA) on cisplatin (CDDP)-induced expres_sion of transient receptor potential vanilloid 1 (TRPV1) in mouse cochlea .Methods Sixty BALB/c mice were ran_domly divided into 4 groups (15 mice in each group) and received introperitoneal injection once daily for 5 days:Control group (normol saline) ,UA group (80 mg/kg/day) ,CDDP group (4 .5 mg/kg/day) ,and CDPP (4 .5 mg/kg/day) plus UA group (80 mg/kg/day) .The expression of TRPV1 in mouse cochlea was determined by immuno_histochemistry ,microscope image analysis and western blot ,and auditory thresholds were evaluated by auditory brainstem response (ABR) measurement .ResuIts The expression of cochlea TRPV1 and ABR threshold shift was significantly increased in the mice treated with CDDP (P< 0 .05) ,as compared with control mice .These effects were prevented by UA treatment (all P<0 .05) .Furthermore ,a linear relationship analysis revealed that the ex_pression of cochlea TRPV1 was significantly correlated with ABR threshold shift(|r|>0 .7 , P<0 .05) .ConcIusion UA effectively attenuated CDDP -induced ototoxicity and improved auditory function through inhibition of TR_PV1 .
7.HMGB1 upregulates P-glycoprotein expression in the brain microvascular endothelial cells of the mouse
Yan CHEN ; Nian YU ; Yuan XIE ; Kang ZHANG ; Qing DI
Journal of Medical Postgraduates 2015;(2):141-145
[Abstract ] Objective High-mobility group box-1 (HMGB1) is abundantly released in the epileptogenic brain tissue , but few reports are seen about the effect of HMGB 1 on the expression of P-glycoprotein ( P-gp) in the vascular endothelial cells of the epi-leptogenic tissue .This study is to explore whether HMGB 1 can regulate P-gp expression in the brain microvascular endothelial cells of the mouse in vitro . Methods Immortalized brain microvascular endothelial bEnd .3 cells of the mouse were cultured in vitro and al-located to different concentration groups ( treated with culture medium containing 10 , 100 , 500 , and 1000 ng/mL HMGB1 for 8 hours), treatment duration groups (treated with culture medium containing 100 ng/mL HMGB1 for 4, 8, 16, 24, and 32 hours), and a control group ( treated with culture medium without HMGB 1 ) .The mRNA expression of P-gp-encoding gene-multidrug resistance gene 1a (mdr1a) was detected by real-time qPCR, and its protein expression determined by Western blot and immunocytochemistry . Results The results of qPCR manifested that the expressions of mdr 1a mRNA were 1.646 ±0.176, 1.777 ±0.135, 1.617 ±0.043, and 1.398 ±0.182 in the 10, 100, 500, and 1000 ng/mL HMGB1 groups, respectively, significantly higher than 1.030 ±0.284 in the control group (P<0.05), and so were those in the 4, 8, 16, 24 h, and 32 h groups (2.655 ±0.112, 2.168 ±0.212, 1.823 ± 0.232, 1.418 ±0.376, and 1.445 ±0.123) than in the control (1.010 ±0.164) (P <0.05).Western blot showed a significant increase in the P-gp protein expression in all the concentration groups (P<0.05) as well as in the 8 h and 16 h treatment duration groups as compared with the control group (P<0.05).Immunocytochemis-try also revealed a higher P-gp expression in the HMGB1-treated than in the control cells (P<0.01). Conclusion HMGB1 can upregu-late the expressions of mdr1a mRNA and P-gp protein in the brain microvascular endothelial cells of the mouse , which may associated with drug resistance of central nervous system diseases , especially that of epilepsy .
8.Influence of subculture cycle on tube plantlet growth of Dendrobium huoshanense and medium composition
Shuhua JIA ; Di WANG ; Yuan GAO ; Yongping CAI ; Yi LIN
Chinese Traditional and Herbal Drugs 1994;0(08):-
Objective To optimize the best subculture cycle through studying the influence of subculture time on tube plantlet growth of Dendrobium huoshanense and the change of medium composition.Methods Height,tiller,fresh weight,and chlorophyll content of the tube plantlet and pH value,water content,and sugar content of the medium were measured after different cycles of the subculture,the cost of culture medium for subculture was calculated as well.Results The height of the tube plantlet increase 282.86%,the tiller increase by 3.5 times,fresh weight reaches its maximum,chlorophyll content of the tube plantlet almost reaches its maximum after 40 d subculture;while water content and sugar content of the medium are decreased to the lowest point,pH value of medium(
9.Pilot study on radiotherapy quality control and quality assurance in Zhejiang province
Yuan ZHU ; Xiaoyun DI ; Shenglin MA ; Zhongnan ZHENG
Chinese Journal of Radiation Oncology 1993;0(03):-
Objective To evaluate the methods of carrying out radiotherapy quality control (QC) and quality assurance (QA) in the whole province. Methods From 1995, radiotherapy quality control center of Zhejiang province (the guiding team consists of specialists in radiation oncology of the province) has carried out a QC and QA program including evaluation of administration, departmental infrastructure, equipment, staff and treatment for 28 centers in the whole province. The regulation and scoring system were designed and first informed to every center, with the 28 centers checked and examined by the guiding team from 1999 to 2000. Results Great variations in equipment and staff were observed among participating centers. Equipment condition was not very satisfactory. Most of the treatment protocols were reasonable except that the indication for radiotherapy was not strict enough in some centers. Conclusions It is feasible for the radiotherapy quality control center to check and examine the department of radiation oncology in the whole province. Good QC and QA is invaluable to standardize the treatment protocol and ensure the radiotherapy quality and also helpful to carry out multi center study in the future.