To establish an economical and reproducible method for the high-quality RNA extraction from pancreas, we isolated total RNA from rat pancreas with TRIzol reagent and liquid nitrogen. In the initial stage, we optimized three influential factors, the way to homogenize pancreas, the time to collect the pancreatic tissue from animals, and the weight of the pancreatic tissue in 1 ml of TRIzol reagent. The RNA quality was determined by detecting total RNA content and its absorbance at 260/280 nm wavelength, visualizing RNA in non-denatured agarose gel and performing RT-PCR of pancreas-specific genes. The A (260)/A (280) ratio of the total RNA extracted by grinding 20-30 mg of rat pancreatic tissue removed from the rats in liquid nitrogen within 1 min and then immersed in 1 ml of the TRIzol Reagent was 1.75-1.89, and the ratio of 28S/18S ribosomal RNA bands was more than 1.8. Furthermore, full length of Pdx1 open-reading frame was amplified with RNA extracted from the grinding group rather than from the conventional group. The RT-PCR products of pancreas-specific genes from both exocrine and endocrine parts of pancreas were successfully derived from the extracted RNA. The results suggested that we successfully provided an economical, fast, and reproducible method to obtain the high-quality and intact RNA from rat pancreas with TRIzol Reagent and liquid nitrogen.