λRNA Internal Standards Quantify Sensitivity and Amplification Efficiency of Mammalian Gene Expression Profiling

Abstract

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) crosshybridization 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 (λ) 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.