Objective： To obtain high quality of mitochondrial DNA （mtDNA） and carry out extra-long PCR （XL-PCR）. Methods： Mitochondria were isolated by differential centrifugation, and membranes were disrupted using 10%SDS...Objective： To obtain high quality of mitochondrial DNA （mtDNA） and carry out extra-long PCR （XL-PCR）. Methods： Mitochondria were isolated by differential centrifugation, and membranes were disrupted using 10%SDS （pH 7.0）. mtDNA was then extracted using phenol and chloroform. Resuits： The mtDNA obtained by using our improved method can be used as effective template for XL-PCR, and total mtDNA （16 kb） can be amplified easily. Conclusion： Our improved method is effective in preparing high quality of mtDNA, which can be used as template for XL-PCR.展开更多
目的观察靶向Bcl-x L的miRNAs在机采血小板储存过程中的表达量变化,结合生物信息学分析探讨miRNA对血小板凋亡的可能影响。方法利用实时荧光定量PCR(qRT-PCR)对储存第1 d、第3 d和第5 d的机采血小板中可能靶向Bcl-x L的miRNAs及Bcl-x L ...目的观察靶向Bcl-x L的miRNAs在机采血小板储存过程中的表达量变化,结合生物信息学分析探讨miRNA对血小板凋亡的可能影响。方法利用实时荧光定量PCR(qRT-PCR)对储存第1 d、第3 d和第5 d的机采血小板中可能靶向Bcl-x L的miRNAs及Bcl-x L mRNA的表达量进行检测。评估高表达的miRNA与Bcl-x L的种子序列匹配度(seed match)、热力学稳定性、保守型及靶向性预测分值,并对miRNA表达与Bcl-x L mRNA变化之间的相关性进行分析。结果在血小板储存过程中,靶向Bcl-x L的hsa-miR-133a,hsa-miR-326,hsa-miR-491-5p,hsa-miR-98表达呈上升趋势(P<0.05),其中hsa-miR-326表达量显著增高(P<0.001);hsa-let-7家族(除hsa-let-7c),hsa-miR-133b,hsa-miR-342-3p,hsa-miR-342-5p表达水平无明显改变(P>0.05);hsa-let-7c,hsa-miR-140-5p,hsa-miR-184未检测到;而Bcl-x L mRNA在储存过程中表达呈下降趋势。hsa-miR-133a的表达变化与Bcl-x L mRNA呈显著负相关,生物信息学分析显示hsa-miR-133a和hsa-miR-326更有可能靶向调控Bcl-x L。结论Bcl-x L是血小板存活的关键调控蛋白,hsa-miR-133a,hsa-miR-326有可能调控Bcl-x L mRNA的表达,从而影响血小板的凋亡。展开更多
基金Supported by the National Natural Science Foundation of China (No. C30171030, C30472175)China Postdoctoral Science Foundation (No. 2004036014)
文摘Objective： To obtain high quality of mitochondrial DNA （mtDNA） and carry out extra-long PCR （XL-PCR）. Methods： Mitochondria were isolated by differential centrifugation, and membranes were disrupted using 10%SDS （pH 7.0）. mtDNA was then extracted using phenol and chloroform. Resuits： The mtDNA obtained by using our improved method can be used as effective template for XL-PCR, and total mtDNA （16 kb） can be amplified easily. Conclusion： Our improved method is effective in preparing high quality of mtDNA, which can be used as template for XL-PCR.
文摘目的观察靶向Bcl-x L的miRNAs在机采血小板储存过程中的表达量变化,结合生物信息学分析探讨miRNA对血小板凋亡的可能影响。方法利用实时荧光定量PCR(qRT-PCR)对储存第1 d、第3 d和第5 d的机采血小板中可能靶向Bcl-x L的miRNAs及Bcl-x L mRNA的表达量进行检测。评估高表达的miRNA与Bcl-x L的种子序列匹配度(seed match)、热力学稳定性、保守型及靶向性预测分值,并对miRNA表达与Bcl-x L mRNA变化之间的相关性进行分析。结果在血小板储存过程中,靶向Bcl-x L的hsa-miR-133a,hsa-miR-326,hsa-miR-491-5p,hsa-miR-98表达呈上升趋势(P<0.05),其中hsa-miR-326表达量显著增高(P<0.001);hsa-let-7家族(除hsa-let-7c),hsa-miR-133b,hsa-miR-342-3p,hsa-miR-342-5p表达水平无明显改变(P>0.05);hsa-let-7c,hsa-miR-140-5p,hsa-miR-184未检测到;而Bcl-x L mRNA在储存过程中表达呈下降趋势。hsa-miR-133a的表达变化与Bcl-x L mRNA呈显著负相关,生物信息学分析显示hsa-miR-133a和hsa-miR-326更有可能靶向调控Bcl-x L。结论Bcl-x L是血小板存活的关键调控蛋白,hsa-miR-133a,hsa-miR-326有可能调控Bcl-x L mRNA的表达,从而影响血小板的凋亡。
