Madison R D, Robinson G A
Duke University Medical Center, Durham, NC, USA.
Biotechniques. 1998 Sep;25(3):504-8, 510, 512, passim. doi: 10.2144/98253rr06.
There is an increasing interest in being able to document simultaneous levels of multiple mRNAs from limited amounts of mammalian tissue. The combination of amplified antisense RNA (aRNA) and reverse Northern blot analysis is one technology that allows the measurement of relative levels of multiple mRNAs. However, potential problems exist with this approach, such as (i) unknown amplification efficiencies and sensitivity of detection, (ii) an inherent 3' bias of amplified products and (iii) cross-hybridization of homologous mRNAs with the gene targets. Each of these potential problems was addressed experimentally by the use of poly(A) RNA internal standards synthesized from lambda phage (lambda) DNA. The results showed detection levels of as few as 10 copies of the poly(A) RNA internal standards. The internal standards aid in the optimization of reaction conditions and also reduce dependence on traditional "housekeeping" genes whose mRNA levels might or might not change. The overall results of these experiments highlight and extend the general usefulness of amplified antisense aRNA and reverse Northern blot analysis to study mRNA expression profiles.
能够记录来自有限量哺乳动物组织的多种mRNA的同时水平，这一需求日益增长。扩增反义RNA（aRNA）与反向Northern印迹分析相结合是一种能够测量多种mRNA相对水平的技术。然而，这种方法存在一些潜在问题，例如：（i）扩增效率和检测灵敏度未知；（ii）扩增产物存在固有的3'偏向性；（iii）同源mRNA与基因靶标的交叉杂交。通过使用从λ噬菌体（lambda）DNA合成的聚腺苷酸（poly(A)）RNA内标，对这些潜在问题逐一进行了实验研究。结果表明，聚腺苷酸RNA内标低至10个拷贝的水平也能被检测到。内标有助于优化反应条件，还减少了对传统“管家”基因的依赖，因为这些基因的mRNA水平可能会变化，也可能不会变化。这些实验的总体结果突出并扩展了扩增反义aRNA和反向Northern印迹分析在研究mRNA表达谱方面的普遍实用性。
