Evaluation of methods for amplification of picogram amounts of total RNA for whole genome expression profiling

ABSTRACT: For more than a decade, microarrays have been a powerful and widely used tool to explore the transcriptome of biological systems. However, the amount of biological material from cell sorting or laser capture microdissection is much too small to perform microarray studies. To address this issue, RNA amplification methods have been developed to generate sufficient targets from picogram amounts of total RNA to perform microarray hybridisation. In this study, four commercial protocols for amplification of picograms amounts of input RNA for microarray expression profiling were evaluated and compared. The quantitative and qualitative performances of the methods were assessed. Microarrays were hybridised with the amplified targets and the amplification protocols were compared with respect to the quality of expression profiles, reproducibility within a concentration range of input RNA, and sensitivity. Four commercial protocols for amplification of picograms amounts of input RNA for microarray expression profiling were evaluated and compared. For each protocol, one RNA amplification was performed from 250 pg, and one from 500 pg of human universal RNA by two operators in two independent laboratories and compared to the amplified aRNA obtained from 2 µg and 100 ng RNA inputs following the standard protocol proposed by Affymetrix. A negative control (amplification without total RNA) and a positive control (if available) were included in each experimental batch. Samples indicating 50, 100, and 1000 pg RNA inputs correspond to 3 additional quantities of total RNA used to synthesise the cDNA target using the nugen protocol for comparison (250, 500 pg + 50, 100, 1000 pg).

ORGANISM(S): Homo sapiens

SUBMITTER: charles decraene 

PROVIDER: E-GEOD-15398 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress