Comparison of mRNA gene expression by RT-PCR and DNA microarray
Comparison of mRNA gene expression by RT-PCR and DNA microarray
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DOI：
10.2144/04364st02
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发表时间：
2004-04-01
期刊：
影响因子：
2.7
通讯作者：
Meyer, RA
中科院分区：
文献类型：
--
作者：
Etienne, W;Meyer, MH;Meyer, RA
很少有研究比较定量mRAA的DNA微阵列的结果，通过逆转录PCR（RT-PCR）。在这项研究中，mRNA收集从愈合的成年和幼年大鼠股骨骨折骨痂骨折后的不同时间。用两种方法测定了10个样品的26个基因。对于RT-PCR，将mRNA逆转录、扩增、印迹、并用P-32标记的内部寡核苷酸探测，并对其进行定量。对于DNA微阵列，将mRNA加工成生物素标记的cRNA，与10个大鼠U34 A微阵列杂交并定量。RT-PCR和微阵列之间一致性的每个基因的相关系数（r）范围为-0.48至+0.93。这种变异使得解释具有基因特异性。具有中等表达水平的基因给出了最高的r值。微阵列软件缺失调用的数量增加以及PCR打印机和微阵列探针位置之间的分离增加都导致一致性降低。微阵列分析表明，通过RT-PCR测量的两个基因的表达水平的地板效应。总之，PCR引物和微阵列探针位置重叠的中等mRNA表达水平可以在这两种方法之间产生良好的一致性。
Few studies have compared the quantification of mRAA by DNA microarray to the results obtained by reverse transcription PCR (RT-PCR). In this study, mRNA was collected from the healing femoral fracture callus of adult and juvenile rats at various times after fracture. Ten samples were measured by both methods for 26 genes. For RT-PCR, mRNA was reverse transcribed, amplified, electrophoresed, blotted, and probed with P-32-labeled internal oligonucleotides, which were quantified. For DNA microarray, the mRNA was processed to biotin-labeled cRNA, hybridized to 10 Affymetrix(R) Rat U34A microarrays, and quantified. Correlation coefficients (r) for each gene for the agreement between RT-PCR and microarray ranged from -0.48 to +0.93. This variation made the interpretation gene-specific. Genes with moderate expression levels gave the highest r values. Increased numbers of absent calls by the microarray software and increased separation between the location of the PCR printers and the microarray probes both led to reduced agreement. Microarray analysis suggested a floor effect in expression levels measured by RT-PCR for two genes. In conclusion, moderate mRNA expression levels with overlap in the location of PCR primers and microarray probes can yield good agreement between these two methods.