摘要
通过对K562细胞基因表达谱芯片杂交影响因素的研究表明,用限制性显示技术[1]制备的cDNA探针长度较均一,适合基因芯片杂交;在42℃条件下含甲酰胺的杂交液中杂交16～20h,可保证样品的有效杂交,并表现出很好的杂交特异性;用RD-PCR或线性PCR对少量样品进行扩增,并用荧光(Cy3或Cy5)标记的通用引物对样品进行标记,可提高芯片检测的灵敏度;一次杂交反应总RNA的用量仅需0.5～10μg,在每cm2约含1000～1500个点的阵列中杂交时,标记样品用量1～2μg为宜;用PCR产物纯化柱对荧光标记产物进行纯化,可大大减小背景荧光,提高信噪比;同一批芯片经同一样品杂交结果的重复性很好,相关系数高达97.8%.
Studies of effect factors on the hybridization of gene expression microarray of K562 cells showed the length of probes prepared by the technology of restriction display PCR was similar,and fit to hybridization of gene chip.When the hybridization time was from 16 h to 20 h under the condition of hybridization solution containing 25%formamide at 42 ℃,the specificity of hybridization was high.The sensitivity of detection could be improved when universal primers labeled by fluorescence (Cy3 or Cy5) were used to label little sample according to restriction display PCR.The effect of directly labeling total RNA was the same as that of labeling mRNA.The amount of total RNA for one hybridization was 0.5～10 μg.To an array of 1 000～1 500 dots per cm2,the amount of labeled sample was about 1～2 μg.Purifying the labeled samples with PCR purification kit can largely reduce the background fluorescence and improve the ratio of signal to noise.For chips of the same batch,the results of hybridization with the same sample could be highly repeated with 97.8%relative coefficient K562 cell.
出处
《生命科学研究》
CAS
CSCD
2004年第2期133-139,共7页
Life Science Research
基金
国家自然科学基金(39880032)
