Properties of signal intensities observed with individual probes of GeneChip Rat Gene 1.0 ST Array, an affymetric microarray system.

For proper evaluation of the results of microarray experiments, it is important to understand how the signal intensities of individual probes are determined. Our previous studies revealed that signal intensities of individual probes in the Agilent array system (code G4131F) are largely dependent upon the location of the probes in the mRNA. In the present study, we examined the properties of signal intensities of individual probes in an Affymetrix array system (GeneChip Rat Gene 1.0 ST Array), in which a random primer fused to the T7 promoter sequence is employed. Distinct from the Agilent array system, individual probes used in this Affymetrix array system did not show the probe-location effects, but gave relatively diverse signal intensities. However, the diversities of the signal intensities of these individual probes were not due to experimental error.

Sequential analysis of RNA synthesis by microchip electrophoresis.

We describe the potential of microchip electrophoresis with a Hitachi SV1100, which can be used to evaluate the integrity of total RNA, for the analysis of synthesized RNA. There was little interference by DNA and/or the components of the in vitro transcription system with the microchip electrophoresis. The fluorescence intensity corresponding to the synthesized RNA increased in a time-dependent manner as to the RNA synthesis reaction on sequential analysis. A result can be obtained in 160 s and only 1/10 aliquots of samples, compared with the conventional method, are required. These results indicate the potential of microchip electrophoresis for sequential analysis of RNA synthesis.

Construction of plasmids suitable for in vitro synthesis of full-length mRNAs having a 3'-poly(A)+tail.

In vitro synthesized mRNAs are often used as calibrants in gene expression analysis. In the case of an analytical procedure for gene expression containing a process of reverse transcription such as microarray analysis, full-length mRNAs having a 3'-poly(A)+tail are required as calibrants. However, the preparation of full-length mRNAs having a 3'-poly(A)+tail by using ordinary plasmid vectors is difficult. In this study, we established two plasmid vectors in which the nucleotide sequence corresponding to the 3'-poly(A)+tail were included. By simply inserting full-length cDNAs lacking their 3'-poly(A)+tail into established plasmid vectors, full-length mRNAs having a 3'-poly(A)+tail were successfully prepared.

Evaluation of cell-free protein synthesis using PDMS-based microreactor arrays.

A living cell has numerous proteins, only a few thousand of which have been identified to date. Cell-free protein synthesis is a useful and promising technique to discover and produce various proteins that might be beneficial for biotechnological, pharmaceutical, and medical applications. For this study, we evaluated the performance and the general applicability of our previously developed microreactor array chip to cell-free protein synthesis by comparisons with a commercially available system. The microreactor array chip comprises a temperature control chip made of glass and a disposable reaction chamber chip made of polydimethylsiloxane (PDMS). For evaluation of the microreactor array chip, rat adipose-type fatty acid binding protein, glyceraldehyde-3-phosphate dehydrogenase, cyclophilin, and firefly luciferase were synthesized from their respective DNA templates using a cell-free extract prepared from Escherichia coli. All these proteins were synthesized in the microreactor array chip, and their respective amounts and yields were investigated quantitatively.

Importance of probe location for quantitative comparison of signal intensities among genes in microarray analysis.

In our previous studies, we demonstrated that expression levels of genes determined by Agilent oligoarray system, code G4130A, could be quantitatively evaluated by spike-in of synthetic full-length mRNAs as standards [Kakuhata R, Watanabe M, Yamamoto T, Akamine R, Yamazaki N, Kataoka M, et al. Possible utilization of in vitro synthesized mRNAs specifically expressed in certain tissues as standards for quantitative evaluation of the results of microarray analysis, J Biochem Biophys Methods 2007;70:755-60]. However, in its successor version (Agilent oligo array system, code G4131F), which was established to enable gene expression analysis over a much wider dynamic range, multiple probes are often utilized for evaluation of expression levels of individual genes; and they showed markedly distinct signal intensities. This result indicates that the observed signal intensities in this new version seemed not to simply reflect the transcript levels of individual genes. To understand the factors influencing the signal intensities of probes, we characterized the properties of the probes used in this new array system and the cRNAs formed during the labeling process. Analysis of cRNAs in the reaction mixture, which were hybridized with the arrays, revealed that the cRNAs were not fully transcribed under the conditions used. For this reason, probes located at the 5' side of the message were found to give lower signals than those at the 3' end; and the observed signal intensities were dependent upon the location of probes in the mRNA. Analysis of the correlation between signal intensities and locations on mRNAs for larger numbers of probes also supported the idea that probe location is one of the major determinants of signal intensities of probes in microarray analysis.

Synchronized changes in transcript levels of genes activating cold exposure-induced thermogenesis in brown adipose tissue of experimental animals.

To identify genes whose expression in brown adipose tissue (BAT) is up- or down-regulated in cold-exposed rats, we performed microarray analysis of RNA samples prepared from the BAT of cold-exposed rats and of rats kept at room temperature. Previously reported elevations of transcript levels of uncoupling protein (UCP1), type II iodothyronine deiodinase (DIO2), and type III adenylate cyclase (AC3) in the BAT of cold-exposed rats over those in that of rats maintained at room temperature were confirmed. In addition to these changes, remarkable elevations of the transcript levels of several genes that seemed to be associated with the processes of cell-cycle regulation and DNA replication were detected in the BAT of cold-exposed rats, possibly reflecting the significant proliferation of brown adipocytes in response to cold exposure. Up-regulation of the gene encoding sarcomeric mitochondrial type creatine kinase in the BAT of cold-exposed rats was also detected by microarray analysis, but subsequent Northern analysis revealed that the expression of not only the sarcomeric mitochondrial type enzyme, but also that of 2 other subtypes, i.e., cytoplasmic brain type and cytoplasmic muscle type, was elevated in the BAT of cold-exposed rats. Microarray analysis also revealed a significant expression of myoglobin in BAT and its elevation in the BAT of cold-exposed rats, and this result was supported by calibrated Northern analysis. On the contrary, several genes such as regulator of G-protein signaling 2 and IMP dehydrogenase 1 were down-regulated in the BAT of cold-exposed rats. The physiological meaning of these changes accompanying cold exposure was discussed.

Possible utilization of in vitro synthesized mRNAs specifically expressed in certain tissues as standards for quantitative evaluation of the results of microarray analysis.

To examine the possible usefulness of in vitro synthesized RNA as standards in microarray analysis, we prepared full-length mRNAs encoded by 3 rat metabolic genes for heart/muscle type carnitine palmitoyltransferase I (M-CPTI), uncoupling protein (UCP1), and heart/muscle type fatty acid-binding protein (H-FABP). Artificial RNA samples were prepared by adding known amounts of these synthetic mRNAs to total RNA from rat liver, and transcript levels of various genes were compared between the prepared artificial RNA samples and total RNA samples of rat liver by using an Agilent oligo microarray system. Upon the addition of these synthetic RNAs, signals from the DNA spots corresponding to these 3 genes were elevated, but those from the DNA spots representing other genes were not markedly influenced. Using the ratio of the increase in signal intensity of DNA spot to the amount of added RNA, we estimated the expression levels of several genes and compared them with the absolute expression levels determined by calibrated Northern analysis. As a result, the absolute transcript levels of 3 genes encoding acidic ribosomal phosphoprotein P0, type-1 voltage-dependent anion channel (VDAC1), and type-2 glucose transporter (GLUT2) were successfully estimated by this procedure. Furthermore, genes specifically expressed in certain tissues such as UCP1 were concluded to be good candidates as standards for use in microarray analysis.

Platelet-derived growth factor stimulates glucose transport in skeletal muscles of transgenic mice specifically expressing platelet-derived growth factor receptor in the muscle, but it does not affect blood glucose levels.

Insulin stimulates the disposal of blood glucose into skeletal muscle and adipose tissues by the translocation of GLUT4 from intracellular pools to the plasma membrane, and consequently the concentration of blood glucose levels decreases rapidly in vivo. Phosphatidylinositol (PI) 3-kinase and Akt play a pivotal role in the stimulation of glucose transport by insulin, but detailed mechanisms are unknown. We and others reported that not only insulin but also platelet-derived growth factor (PDGF) and epidermal growth factor facilitate glucose uptake through GLUT4 translocation by activation of PI 3-kinase and Akt in cultured cells. However, opposite results were also reported. We generated transgenic mice that specifically express the PDGF receptor in skeletal muscle. In these mice, PDGF stimulated glucose transport into skeletal muscle in vitro and in vivo. Thus, PDGF apparently shares with insulin some of the signaling molecules needed for the stimulation of glucose transport. The degree of glucose uptake in vivo reached approximately 60% of that by insulin injection in skeletal muscle, but blood glucose levels were not decreased by PDGF in these mice. Therefore, PDGF-induced disposal of blood glucose into skeletal muscle is insufficient for rapid decrease of blood glucose levels.