In this study, we developed a simple and reliable method, Gene Expression Profiling via Multigene Concatemers (MgC-GEP), for multigene expression profiling. We characterized the gene transcription of 20 genes in Saccharomyces cerevisiae that was under the weak acid stress using MgC-GEP and validated the results by real time RT-PCR. Our results show that MgC-GEP is a powerful gene expression profiling tool for a moderate number of multi- genes to search and identify key players in the particular pathways of interest. Materials and Methods Strains and Vectors S. cerevisiae strain W303-1A was obtained from CICIM-CU in Jiangnan University of China. The pUC19 T-vector (TaKaRa, China) was used to clone the concatemers. Escherichia coli strain JM109 was used for routine bacterial transformations and maintenance of plasmids. RNA extraction Samples were prepared as previously described [40]. Yeast cells from overnight cultures in yeast peptone dextrose (YPD) medium were diluted in fresh YPD to an OD600 of 0.1 and grown at 30uC until an OD600 of 1–1.1 was reached. Cultures were split and then potassium sorbate (BBI, USA) was added at a final concentration of 8 mM to one half of the culture. After 20 min, both untreated and treated cultures were harvested by centrifu- gation at room temperature (2 min, 40006g), and cells were immediately washed in ice-cold water, reharvested at 4uC, snap- frozen in liquid nitrogen and stored at 280uC. Total RNA was prepared using an SV Total RNA Isolation System (Promega, USA) according to the manufacturer’s instructions. Residual DNA was digested with RNase-free DNase (DNase I, TaKaRa, China) at 37uC for 30 min. After heat inactivation for 10 min at 65uC in 2 mM EDTA, 1.5 ml total RNA solution was removed for quantification. Gene Expression Profiling via Multigene Concatemers (MgC-GEP) According to the principles of GenomeLabTM GeXP Genetic Analysis System, we designed 20 pairs of multiplex gene-specific primer with a tag for the 20 genes in S. cerevisiae strain W303-1A and 1 pair of universal primer by using Beacon Designer 2.0 software (Bio-Rad, USA) (Table S1). Forward gene-specific primers consisted of 16–20 nucleotides corresponding to the target gene coupled to an 18-nucleotide universal forward tag sequence with a BamHI site. Reverse gene-specific primers consisted of 16–20 nucleotides complementary to the target gene coupled to a 19-nucleotide universal reverse tag sequence with HindIII site. The pair of universal primers is the 18-nucleotide universal forward tag sequence and the 19-nucleotide universal reverse tag sequence. The average Tm of all 20 gene-specific primers is 67.361.8uC with difference from each other within 5uC. And PCR products are 8567 bp in length. The amplification efficiencies of specific primers were examined using real-time RT-PCR (Table S3). For each sample, reverse transcription (RT) was followed by PCR (Fig. 1). RT reactions mixtures (25 ml) contained 500 ng RNA, 0.16 mM reverse gene-specific primer mix, 200 U reverse transcrip- tase (Promega, USA), 25 U RNase inhibitor (TaKaRa, China), 5 ml M-MLV 56reaction Buffer, and 0.5 mM each dNTP. RT reactions were incubated at 70uC for 5 min, 42uC for 60 min. Subsequent PCR was done with each reaction containing 2 ml RT reaction, 0.02 mM forward primer set mix, 2.5 U Taq DNA polymerase Multigene Expression Profiling (Bioflux, Japan), 0.2 mM each dNTP, and 106PCR buffer (Bioflux, Japan) containing 5 mM MgCl2, 10 mM Tris-HCl, 50 mM KCl, 1 mM universal forward primer, and 1 mM universal reverse primer. Amplification conditions consisted of initial denaturation at 95uC for 5 min, followed by 28 cycles of 94uC for 30 s and 53uC for 30 s, 72uC for 35 s ending in a single extension cycle of 72uC for 5 min. The PCR product was extracted with PC8 (phenol:chloroform 1:1, pH 8.0) and precipitated with isopropanol, and dissolved in Lo TE buffer (3 mM Tris-HCl, pH 7.5; 0.2 mM EDTA, pH 7.5) after washed by 70% ethanol. The purified and concentrated product was digested by HindIII and BamHI for 12 h and treated by chloroform extraction and isopropanol precipitation, and dissolved in Tris- EDTA (TE) buffer. The digested product was separated by 3% agarose gel electrophoresis. The gel containing of digested target bands was cut and centrifuged by spin column. The product was precipitated by ethanol and dissolved in Lo TE buffer. The digested products were ligated to produce concatemers by 5 U T4 DNA ligase (TaKaRa, China) and 15% PEG6000 at 16uC for 4 h. The concatemers were separated by 1.5% agarose gel electrophoresis. The region 500 bp–1200 bp was isolated and cloned in the pUC19T-vector digested by HindIII and BamHI. Finally, the recombinant plasmids were transformed into E. coli competent cells. We randomly selected 300 clones from each sample and inserts were sequenced using ABI PRISM 3730 automatic DNA sequence systems (SANGON Sequencing Service, Shanghai China). The sequencing primers were M13-479 (59-CGCCAGGGTTTTCC- CAGTCACGAC-39) and RV-M (59-GAGCGGATAACAATTT- CACACAGG-39). The sequence and occurrence of each gene were determined and the Act1 gene encoding Act1p, which was a ubiquitous protein involved in the filament formation, was as the internal control. The sequences were analyzed by using the local BLAST program. All samples were analyzed in triplicate. Real-time RT-PCR analysis For real-time RT-PCR, the cDNA was synthesized from 1 mg of DNaseI-treated total RNA with the anchored oligo-dT primer following the manufacturer’s protocol (Promega, USA). Twenty pairs of specific primers for the 20 genes in S. cerevisiae strain W303- 1A were designed using Beacon Designer 2.0 software (Bio-Rad, USA) (Table S2). Real-time RT-PCR was performed using the SYBR-Green PCR Master Mix kit (Bio-Rad, USA) in the Light Cycler (Bio-Rad, USA). A serial dilution of the cDNA was used as standard curve to optimize the amplification efficiency with each primer pair. Three technical replicates were run for each of the standards. The cycling conditions were 95uC for 15 sec followed by 40 cycles of 95uC for 15 sec, 53.5uC for 15 sec and 72uC for 15 sec. Amplification specificity was confirmed by generating a melting curve of the PCR products. The amplified fragments were also verified by gel electrophoresis. Act1, which encoded Act1p, a ubiquitous protein involved in the filament formation, was amplified as the internal control. Relative target gene expression was determined with the comparative cycle threshold (CT) method. The DCT value was calculated by subtracting the target CT for each sample from its Act1 CT value. Every RT-PCR experiment was repeated with three biological samples and each sample was run in triplicate. The relative expression level of mRNA was analyzed between the weak acid-induced samples and non-induced samples with DPS statistical analysis software. Results Scheme of MgC-GEP A schematic diagram of the technique is shown as Fig. 1. First, total RNA extracted from samples was reverse transcribed using PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e15711