Objective Based on real-time PCR technique and ring primers,to establish a simple,accurate,cost-effective and easily standardized quantitative assay for quantification of HER2 mRNA,and apply to provide medication guidance for clinical tumor personalized molecular targeted therapy.Methods Screening reference gene which was stable expression in breast cancer,and optimizing the PCR reaction system.Then a real-time PCR with Eva Green for quantification of the mRNA expression levels of HER2 gene was developed.The specificity,sensitivity and reproducibility of the method were evaluated 87 specimens including 55 liquid nitrogen-frozen breast cancer tissues and 32 normal tissues were detected by the real-time quantitative reverse transcription (FQ RT)-PCR and immunohistochemistry(IHC).Results The standard curve of the method indicated a good linear relationship between the Ct value and the template concentration with the correlation coefficient being 0.997.The linear range of the system was from 101 to 106 copies/μl and the lower detection limit was 101 copies/μl.It had a high sensitivity and good specificity.The inter-assay coefficients of variation of HER and RPL37A genes were (5.93 ± 0.57)％ and (5.11 ± 0.59)％,(2.49 ±0.81)％ and (2.98 ±0.97)％ respectively.The intra-assay coefficients of variation were (5.76 ±0.58)％and (7.71 ±0.61)％,(3.75 ±0.76)％ and (4.40 ±0.96)％ respectively.Using the optimized FQ RTPCR system,HER2 gene of 87 specimens was quantificated.The sensitivity of the assay was 96.36％ (53/55),the specificity was 78.13％ (25/32),the positive predictive value was 88.33％ (53/60),the negative predictive value was 92.59％ (25/27),and the total coincidence rate between FQ RT-PCR and IHC was 89.66％ (78/87).The correlation of the results between the FQ RT-PCR and IHC was good (Kappa =0.770,P ＞ 0.05).Conclusions The method can quantify the mRNA expression levels of HER2 gene rapidly and cost-effictively with high sensitivity and specificity.It can be applied to clinical molecular diagnosis with attractive prospect.

Purpose Sustained attention dysfunction is a common symptom of patient with attention-deficit/hyperactivity disorder (ADHD).To reveal the neural mechanism of the abnormality of sustained attention of patients with ADHD,the cerebral blood flow (CBF) abnormalities in patients were studied by perfusion imaging.Materials and Methods Sixteen children with ADHD and twenty normal controls treated at the 401st Hospital of PLA from June 2013 to September 2015 underwent 3D arterial spin labeling (3D-ASL)scanning with GE 3.0T MRI scanner.The participants were performed four continuous sections of sustained attention to response task (SART) for 20 minutes in the scanner.Using SPM 8 toolkit,the local CBF values of both groups were compared in a voxel-wise manner,and their correlations with response time and target accuracy of SART were analyzed.Results When performing the SART,the patients with ADHD showed significantly inhibited trend of increasing CBF in the anterior cortex like dorsal cortex,medial prefrontal cortex,and motor area;however,they presented enhanced trend of increasing CBF in the posterior cortex such as posterior cingulate cortex and parietooccipital sulcus (P＜0.01);the change of CBF in the dorsal prefrontal cortex and that in the precentral and postcentral gyrus had significant correlation with response time of SART task and targeting ratio (dorsal prefrontal cortex:r=0.745,P＜0.001;r=0.591,P＜0.001;r=-0.521,P＜0.001.Precentral and postcentral gyrus:r=0.579,P＜0.001).Conclusion Patients with ADHD show different CBF redistribution between anterior and posterior cerebral cortex in performing SART,and the abnormal CBF shows significant correlations with behavioral metrics,which reflects the mechanism of sustained attention dysfunction of patients with ADHD.