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cDNA微阵列制作的优化
引用本文:黄宝俊,赵雨杰,徐惠绵,何群,张玉魁,徐莹莹,马佳明HUANG Bao-Jun,ZHAO Yu-Jie,XU Hui-Mian,HE Qun,ZHANG Yu-Kui,XU Ying-Ying,MA Jia-Ming.cDNA微阵列制作的优化[J].遗传,2003,25(5):691-595.
作者姓名:黄宝俊  赵雨杰  徐惠绵  何群  张玉魁  徐莹莹  马佳明HUANG Bao-Jun  ZHAO Yu-Jie  XU Hui-Mian  HE Qun  ZHANG Yu-Kui  XU Ying-Ying  MA Jia-Ming
作者单位:1.中国医科大学附属第一医院肿瘤外科,沈阳 110001; 2.中国医科大学生物芯片中心,沈阳 110001 1.Oncology Department,The First Affiliated Hospital,China Medical University,Shenyang 110001,China; 2.The Center of Biochip,China Medical University,Shenyang 110001,China
摘    要:为了优化筛检cDNA微阵列中靶基因的最适长度、浓度及点样溶液的种类,设计持家基因beta actin和GAPDH RT-PCR 3对引物,产物长度在189~1 078 bp之间,以乙肝病毒DNA片段为阴性对照,扩增纯化后分别溶于3×SSC、50%DMSO及0.5mol/L碳酸盐缓冲液(pH=9.0)中,调整浓度分别为0.5μg/μL、1.0μg/μL和1.5μg/μL,比较上述不同条件的杂交结果。结果表明,杂交具有较好的特异性,阴性对照(乙肝病毒)和空白对照(点样溶液)均未见杂交信号;3种长度的同一靶基因杂交信号强度无明显差别(beta actin P=0.378;GAPDH P=0.866);3种点样溶液中以50%DMSO杂交信号最好,较强且均匀一致(P=0.0001),其余2种差异不显著(P=0.142);3种浓度靶基因杂交信号差异不显著(P=0.648),浓度高者信号略强。短片段靶基因(200 bp左右)可获得与长片段靶基因(1 000 bp以上)一样较好的杂交信号,点样溶液以50%DMSO效果最好,靶基因浓度为0.5μg/μL时即可得到较好的杂交结果。 Abstract:To optimize and screen the most suitable target gene length,concentration and printing solution in cDNA microarray,housekeeping genes,such as beta actin and GAPDH,were selected as targets and hepatitis B virus gene as negative control.The RT-PCR primers that spanned at least one intron and whose products were at between 189 bp and 1 078 bp were designed with primer premier 5.0,so did the hepatitis B virus gene PCR primer.After polymerase chain reaction,the products were purified with ethanol and dissolved in 3×SSC,50% DMSO and 0.5mol/L carbonate buffer(pH=9.0)respectively.The concentrations of target genes were adjusted at 0.5μg/μL,1.0μg/μL and 1.5μg/μL.The hybridization signals had a good specificity.No signal showed in either negative control (HBV) or blank control (printing solution only).There was no significant difference in target gene lengths.The P value of beta actin (189 bp,491 bp,974 bp) and GAPDH (227 bp,552 bp,1 078 bp) was 0.378 and 0.866 respectively.There was no significant difference among concentrations(P=0.648),too.However,the higher the concentration was,the stronger the signals would be.Among the three kinds of printing solution,50% DMSO was the best(P=0.0001),while the other two had no difference by multi-comparison(P=0.142).The target gene at length between 200 bp and 1 000 bp has got the same hybridization signals.50%DMSO printing solution and the target gene concentration of 0.5μg/μl are suitable for good hybridization.

关 键 词:点样溶液  Key  words  cDNA  microarray  target  gene  cDNA微阵列  靶基因  

Optimization of cDNA Microarray Fabrication
HUANG Bao-Jun,ZHAO Yu-Jie,XU Hui-Mian,HE Qun,ZHANG Yu-Kui,XU Ying-Ying,MA Jia-Ming.Optimization of cDNA Microarray Fabrication[J].Hereditas,2003,25(5):691-595.
Authors:HUANG Bao-Jun  ZHAO Yu-Jie  XU Hui-Mian  HE Qun  ZHANG Yu-Kui  XU Ying-Ying  MA Jia-Ming
Abstract:
Keywords:
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