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A method for gene expression analysis by oligonucleotide arrays from minute biological materials

2004; Elsevier BV; Volume: 331; Issue: 2 Linguagem: Inglês

10.1016/j.ab.2004.03.039

1096-0309

Wan Ji, Wenli Zhou, Keqin Gregg, Klaus Lindpaintner, Sara K. Davis, Scott Davis,

Advanced biosensing and bioanalysis techniques

Gene expression profiling has been widely used in identifying differentially expressed genes. One of the most popular formats is oligonucleotide array. A limitation of oligonucleotide arrays is the requirement of relatively large amounts of biological starting materials for gene expression analysis. We have developed a simple method for gene expression profiling from very small amounts of biological material by combining exponential (PCR) and linear (T7 RNA polymerase) amplification. By modifying the widely used SMART protocol, we combined T7 promoter ligation and PCR amplification in one step and generated around 0.5 microg of PCRcDNA from 30 ng of total RNA in a single PCR. The PCRcDNA was in vitro transcribed by T7 RNA polymerase to generate complementary RNA (cRNA), which then was used to hybridize Affymetrix GeneChips. Our results demonstrated a linear correlation between the PCR amplification and the conventional linear amplification in gene expression ratios of individual transcript species between two different RNA preparations. The method was further validated by TaqMan reactions. Moreover, both linear and PCR methods showed some inherent bias as to which transcripts were detected, suggesting that using both in parallel may provide a more comprehensive coverage of the transcriptome present in a given sample.