Objective To detect traces of microRNAs (miRNAs) in plasma and assess the expression stability of two common reference genes by stem-loop and poly A polymerase (PAP) real-time quantitative polymerase chain reaction (PCR) method, as miRNAs are the new bio-markers of tumor diagnosis and molecular targeted therapy, and its quantitative research is very important. Methods We extracted miRNAs from plasma of adult Sprague Dawley (SD) rats’ plasma, and detected the expressions of rno-miR-200b-3p and rno-miR-126-3p with stem-loop and PAP real-time PCR quantitative method, with rno-miR-103a-3p and U6 as internal controls. All the results were evaluated by 2△Ct method. Results Compared with PAP method, the stem-loop method reduced Ct value by 2-4 cycles and improved sensitivity by 10 times. In PAP method, the melting curve showed two peaks, a main peak and a small non-specific peak. Yet the melting curve of stem-loop method demonstrated a single specific peak. Furthermore, we validated the stability of internal references in the two real time PCR methods. U6 presented a more stable Ct value than rno-miR-103 in adult SD rats’ plasma samples. Conclusions Stem-loop real-time PCR is recommended as a major way to detect some samples with a low concentration of miRNAs, owing to its high accuracy and sensitivity. However, if a large number of tissue samples is going to be detected, PAP real-time PCR is more suitable and convenient than stem-loop method. U6 is more stable and repeatable than rno-miR-103a-3p as the reference gene to evaluate the semi-quantitative consequence of miRNAs.
ObjectiveTo compare four different transfection reagents for transfection efficiency of rat heart myoblast cells H9C2, to choose the optimal transfection method.MethodsThe plasmids of enhanced green fluorescent protein (EGFP) gene were transfected as exogenous genes to H9C2 cells from four different transfection regents including FuGENE HD, DNA-In CRISPR, Lipofectamine 3000 and Lipofectamine 2000. Fluorescence intensity was measured by fluorescence microscopy and fluorescence microplate reader to evaluate transfection efficiency. The effects of four transfection reagents on cell viability were measured by Cell Counting Kit-8 (CCK-8) reagents.ResultsTransfection efficiency of Lipofectamine 3000 was the highest (>50%), while that of DNA-In CRISPR was the lowest (<1%). The cytotoxicity of Lipofectamine 3000 was the lowest in the four transfection reagents and the cell viability was 94.55% after 48-hour transfection.ConclusionTransfection regent Lipofectamine 3000 has the relatively high transfection efficiency as well as the lowest cytotoxicity, which is more suitable for use in H9C2 cells by transfection.
目的 通过分析环境空气中二氧化硫与过敏原的关联性，探讨二氧化硫在评估过敏性疾患中的作用。 方法 收集2005年1月1日－2012年12月31日绵阳市4个监测点二氧化硫浓度，及2007年7月1日－2012年12月31日的过敏性疾病患者各种过敏原的阳性百分比，采用Pearson相关分析探讨二氧化硫浓度变化对过敏原的影响。 结果 2005年－2012年绵阳市二氧化硫年平均浓度分别为（0.060 ± 0.022）、（0.054 ± 0.018）、（0.046 ± 0.012）、（0.030 ± 0.020）、（0.026 ± 0.010）、（0.035 ± 0.012）、（0.036 ± 0.008）、（0.030 ± 0.009） mg/m3。淡水鱼组合（fs34），海鱼组合（fs33），羊肉（f88），黄豆（f14），花生（f13），狗上皮（e2）与二氧化硫的Pearson相关系数分别为：0.144、0.186、0.209、0.150、0153、0.197。检验P值分别为：0.019、0.002、0.001、0.015、0.013、0.001。按α=0.05标准认为与二氧化硫存在正相关关系。而其余的尘螨组合（ds1）、普通豚草（w1）、律草（u80）、树组合（ts20）、雷菌组合（ms1）、蟑螂（i6）、屋尘（h1）、牛肉（f27）、虾（f24）、蟹（f23）、牛奶（f2）、鸡蛋白（f1）、猫毛（e1）、艾蒿（w6），按α=0.05标准，不能认为与二氧化硫存在正相关关系。 结论 环境空气二氧化硫与淡水鱼、海鲜、羊肉、大豆、花生及狗皮屑具有明确的相关性，对树组合、普通豚草、艾蒿、尘螨组合、屋尘、猫毛、蟑螂、霉菌组合、律草、鸡蛋白、牛奶、牛肉、虾、蟹等无明确的相关性。
Objective To observe the effects of hydrogen peroxide on the expression of transforming growth factorβ1(TGF-β1) and Smad3 protein in A549 cells. Methods A549 cells were cultured with different concentrations of hydrogen peroxide. MTT assay was used to determine the cell growth and survival rates. The level of TGF-β1 and p-Smad3 protein were detected by western blotting. Results It was observed that hydrogen peroxide significantly inhibit proliferation of A549 cells. When the concentration of hydrogen peroxide was 1.0 mmol/L, the inhibition ratio reaches 46.34%, and the level of TGF-β1 and p-Smad3 protein were increased in a time-dependence manner and reached a peak after 24 h, then decreased a little but also remained at high level. Conclusions In the early oxidative damage, A549 cells express high level of TGF-β1 and p-Smad3 protein. It may be relevant to tissue repair and remodeling after lung injury.