Simultaneous Comparison of Gene Expression levels between six different sources by source-specific　adapter mediated PCR and pyrosequencing

摘要

To globally understand the gene function, an efficient method to comparatively detect the expression levels of a given gene at different tissue or at different state is required. Here we describe a novel method for gene expression profiling between six different sources. This method includes five steps: (1) Extract total RNA from different tissues or cells and followed by a reverse-transcription into double-stranded cDNA (ds-cDNA)；(2) Digest the ds-cDNA with restriction endonucleases Mbo I; (3) Label the digested ds-cDNA fragments with source-specific adapters by T4 ligase; (4) Amplify the ligated fragments with a universal primer and a gene specific primer; (5) Decode the sequence of the source-specific area in the amplicons by pyrosequencing. The sequence in the pyrogram represents the source of the gene, and the intensity of the peak represents the relative expression level. The signal ratio reflects the proportion of the amount of mRNAs between different sources. The GAPDH gene and the β-actin gene in mouse Mus tissue were employed for evaluating the method. The relative expression level of the GAPDH gene and the β-actin gene between six identical sources was determined as 0.94:0.93:1.04:0.94:1.06:1.09, and 0.92:0.94:1.05:0.91:0.97:1.2 respectively. These results are very close to the theoretical ratio of 1:1:1:1:1:1; indicating that the method is very accurate for comparing the gene expression level between multiple sources.