Establishment of a rapid procedure for one-step real-time reverse transcription-polymerase chain reaction in combination with automated mRNA extraction: a clinical application to intra-operative molecular diagnosis of cancer cells

Abstract

Reverse transcription-polymerase chain reaction (RT-PCR) technique is extremely useful for sensitive detection of cancer cells, but it requires at least 8 h. If the precise information on the cancer spread based on RT-PCR is available during surgery, we can give the optimal therapy to each patient during operation. We aimed to establish the ultra-rapid procedure of RT-PCR for application to the intra-operative diagnosis. We have established a one-step real-time RT-PCR using LightCycler with hybridization probes, in combination with automated mRNA extraction on MagNA Pure LC. The method can be completed in only 70 min after sampling and has a comparable sensitivity to conventional RT-PCR and/or two-step real-time RT-PCR with a wide (10–10 6) dynamic range. When this method was applied to the intra-operative detection of free cancer cells in peritoneal lavage, the results were available during operation in all cases. We could detect cancer cells more effectively compared to conventional cytology and RT-PCR. This protocol is easily accomplished, delivers rapid, sensitive and reproducible results, and is therefore applicable to intra-operative diagnosis.