Quantitative gene expression analysis in renal biopsies: a novel protocol for a high-throughput multicenter application.

Abstract

Recent advances in gene expression analysis may add the quantification of mRNA species in renal biopsies to routine diagnostic procedures in nephrology. A systematic evaluation was performed on the relevant steps required to efficiently obtain cDNA from renal biopsies for high-throughput reverse transcription-polymerase chain reaction (RT-PCR) based mRNA quantification. The protocol preserves mRNA integrity by a novel RNase inhibitor and allows meticulous microdissection followed by maximal RNA recovery from tissue samples. Reverse transcription was optimized to give the best yield from minimal starting material. RNA quantity and quality were systematically investigated by real-time RT-PCR and electrophoresis on a microfluidic system, respectively. The reported procedure offers high RNA preservation and increases the yield of cDNA significantly compared to former protocols. The simplicity of biopsy material acquisition combined with the centrally performed processing makes this protocol suitable for a wide spectrum of expression analysis in diverse clinical settings.