Insulin gene expression levels can be detected and quantified using a broad range of molecular biology techniques. Traditionally, detection of insulin mRNA levels in both β-cell lines and purified islets has been performed by Northern blot analysis, using the full-length insulin cDNA as a labeled probe (1). This technique is described here, and is still the most useful method for detection of endogenous insulin mRNA levels (densitometry being applied to allow quantification of band intensities). A more rapid analysis of endogenous mRNA levels can be performed by reverse transcriptase-polymerase chain reaction (RT-PCR), which is also described here. RT-PCR allows analysis of insulin mRNA levels from even small amounts of cells or tissue and can be used to analyze expression in as little as a single islet. Like Northern blotting, prior purification of high-quality RNA is essential for this technique and there are several commercial RNA preparation kits available (e.g., RNeasy from Qiagen). Such commercially available kits can also be used to isolate poly-A+ RNA (mRNA) only, which assists in decreasing background signals. To investigate both rapid and long-term effects on insulin gene expression, advances in reporter gene technology have allowed the development of synthetic constructs containing the insulin gene promoter. When transiently transfected into β-cells and β-cell lines, these can now be used to estimate changes in insulin gene expression through measurement of insulin gene promoter activation. Insulin-promoter reporter gene constructs allow rapid and easily quantifiable analysis of expression in a range of cell lines, and in response to external cues, they have become increasingly popular in the analysis of insulin gene expression.
|Title of host publication||Diabetes Mellitus|
|Subtitle of host publication||Methods and protocols|
|Number of pages||9|
|Publication status||Published - 2003|