cDNA microarray analysis after laser microdissection in proliferating islets of partially pancreatectomized mice

Abstract

With islet transplantation having grown in popularity since the introduction of the Edmonton protocol, how to secure an unlimited source of islets has become an urgent problem. To resolve this problem, techniques to induce or proliferate islets are urgently required. To achieve this goal, gene expression analysis using a cDNA microarray in islets of partially pancreatectomized mice, in which the remaining islets regenerate and proliferate with insulin secretion and glucose responsiveness, provides us with valuable information. However, those experiments have two critical problems: first, how to selectively collect the regenerating or proliferating islets, and second, the shortage of total RNA extracted from one islet for a microarray analysis. A useful system was thus designed which combined laser microdissection, cDNA amplification by SMART PCR, which can maintain the relative expression profile of transcripts throughout reactions, and a cDNA microarray. Furthermore, this system is expected to contribute to future studies regarding not only islet regeneration but also the function of the islet itself, and this system may also be applicable to many other types of endocrine tissue. In this review, the details of this system are presented and discussed.

Global expression analysis of the brown alga Ectocarpus siliculosus (Phaeophyceae) reveals large-scale reprogramming of the transcriptome in response to abiotic stress

“…This demonstrated that the protocol for cDNA amplification provided reliable measures of the relative transcript abundances. Although this method has been successfully applied in several small-scale expression studies [ 29 - 35 ], to our knowledge, the use of this technique has not been reported with commercial photolithographically synthesized arrays.…”

Laser-assisted microdissection for real-time PCR sample preparation

“…RNA amplification have been extensively used by several groups (Klur et al, 2004;Wu et al, 2006;Murphy et al, 2005;Coco et al, 2005;Schlomm et al, 2005;Katsuta et al, 2005) when combining LMD and microarray hybridization to identify characteristic genes or gene expression patterns crucial to the understanding of tissue function in normal and diseased states, because the RNA amount isolated from the microdissected specimens (usually in the pg or low ng range) is not sufficient for microarray analysis (several lg are required). Now RNA amplification systems are emerging as method for preparing amplified cDNA from total RNA for gene expression analysis using real-time quantitative PCR (Kurn et al, 2005) for global gene expression analysis from very small samples.…”

β-Cell regeneration: the pancreatic intrinsic faculty

“…Both processes can occur, nevertheless, in absence of 'true' islet regeneration. Among these factors, some endogenous proteins were identified by high-throughput transcriptome and proteome screens of regenerating pancreata [87][88][89][90]. Whole pancreatic extracts after injury appear to promote b-cell mass augmentation in mice treated with STZ [91,92].…”