Multigene Expression Profiling Figure 1. Scheme of MgC-GEP (see text for details). doi:10.1371/journal.pone.0015711.g001 20 reverse primers each containing 19–22 nucleotides comple- mentary to the target gene coupled with a 19-nucleotide universal reverse sequence to obtain the cDNA. The cDNA was PCR- amplified during in the initial 1–3 cycles using the specific primers to synthesize the dozens of gene-amplification products, each of which contained universal tags with specific restriction digestion sites for enzymes a and b at both termini. In this study, the universal tags included the specific restriction digestion sites of BamHI and HindIII. Subsequent PCR amplifications (cycles 4–28) used universal forward and reverse primers to yield amplification products corresponding to each of the 20 specific genes. These two steps effectively condensed mRNA and reduced nonspecific amplifications. The tags corresponding to each of the 20 specific genes were digested with restriction enzyme a and b. The digested products were ligated to produce concatemers with T4 DNA ligase. The concatemers were cloned into the vector digested with restriction enzyme a and b for sequencing. Validation of MgC-GEP To identify potentially protective genes induced by sorbate, the whole-genome transcriptional profile has been studied by whole- genome microarrays [40]. According to the results of the study, we chose to characterize the gene transcription of 20 genes including 10 up-regulated genes (YPL122C, YNR030W, YDR343C, YGR088W, YPR149W, YCL040W, YBR054W, YNR001C, YDR533C, YDL222C), 9 down-regulated genes (YML123C, YEL046C, YLR180W, YLR355C, YLR419W, YLR300W, YNL300W, YLR372W, YAL059W) and Act1 as the internal control in S. cerevisiae that were under the weak acid stress to evaluate the reliability of MgC-GEP conveniently. The target genes were amplified via multiplex RT-PCR (lanes 1 in Fig. 2A) and then the RT-PCR products digested by BamHI/HindIII (lanes 2 in Fig. 2A). After that, the digested 60–70-bp tags were ligated to produce concatemers and 500- to 1200-bp concatemers were collected (Fig. 2B) and cloned into the pUC19 T-vector (TaKaRa, China) digested by BamHI/HindIII. Insert size of 300 clones selected randomly was estimated by PCR analysis (Fig. 2C). The size of the inserts varied from 500 to 1200 bp in length, with an average length of 800 bp. This result demonstrated that there were at least 10 tags in one clone. Inserts were sequenced using ABI PRISM 3730 automatic DNA sequence systems (Sangon, China). The sequence and occurrence of each gene were determined manually and gene expression analysis was performed using the BLAST program. Using MgC-GEP, we found that the expression patterns of 16 genes in 20 selected genes were consistent with the results of previously reported microarray analysis [40]. Seven genes (YNR030W, YDR343C, YGR088W, YBR054W, YNR001C, YDR533C, YDL222C) were up-regulated with weak acid induction and nine genes (YML123C, YEL046C, YLR180W, YLR355C, YLR419W, YLR300W, YNL300W, YLR372W, YAL059W) that were repressed (Fig. 3). Notably, three genes (YPL122C, YCL040W and YPR149W) were proven to be up-regulated during weak acid induction using microarray analysis [40]. However, in MgC-GEP analysis, YPL122C and YCL040W were down-regulated and the transcription level of YPR149W did not noticeably change during weak acid induction. To validate MgC-GEP, we also detected the transcription of the 20 selected genes using real time RT-PCR. The results showed that the expression patterns of 20 genes analyzed by real time RT-PCR were consistent with the results of MgC-GEP analysis. These results demonstrated that MgC-GEP can accurately determine the transcript levels of at least 20 genes at once. Discussion In this study, we present a novel method for multigene expression profiling, designated as MgC-GEP. To validate MgC- GEP, we identified the transcript levels of 20 genes stimulated with PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e15711