Ensuring good quality rna for quantitative real-time pcr isolated from renal proximal tubular cells using laser capture microdissection

摘要: In order to provide gene expression profiles of different cell types, the primary step is isolate specific cells interest via laser capture microdissection (LCM), followed by extraction good quality total RNA sufficient for quantitative real-time polymerase chain reaction (qPCR) analysis. This LCM-qPCR strategy has allowed numerous studies on populations, providing valuable insights into cellular changes in diseases. However, such imposed challenges as interests are often available limited quantities and may be compromised during long periods time spent collection RNA; therefore, it crucial that protocols sample preparation should optimised according populations. We made several modifications existing improve yield integrity downstream qPCR analyses. A modified condensed hematoxylin eosin (H&E) staining protocol was developed identification rat renal proximal tubular (PTCs). It then determined a minimal eight thousands PTCs were required meet qPCR. assessed using at every progressive preparation. Therefore, we decided shortened H&E staining, together with performed consecutively within twenty minutes These later applied six individual samples. panel drug transporters five housekeeping genes showed Ct values below thirty-five, confirming levels these can detected. had successfully optimized achieve from microdissected profiling suitable researchers who interested employing similar applications studies.