Evaluation of Global RNA Amplification and Its Use for High-Throughput Transcript Analysis of Laser-Microdissected Endosperm
作者： Robert C. Day , Les McNoe , Richard C. Macknight
DOI: 10.1155/2007/61028
关键词:
摘要: Laser microdissection (LM) provides a useful method for isolating specific cells or tissues from biological samples. Here, we adapted protocols to allow high-resolution transcript analysis of different developing Arabidopsis seed. Sufficient RNA (∼50 ng) was extracted endosperm tissue RT-PCR. However, obtain enough microarray analyses, it necessary amplify the RNA. PCR- and IVT-based amplification methods were investigated several important technical aspects identified (such as target truncation alterations in signal intensity). We found that when starting only 50 ng RNA, based on PCR IVT produced sufficient product reliable hybridizations, with two-round giving best results. Microarray using endosperm-derived amplified by IVT, reproducibly enriched marker genes. Thus, combined RNA-amplification protocols, LM is robust technique high-throughput tissue-specific gene expression analysis.
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