Chougoni Kranthi Kumar, Grossman Steven R
C. Kenneth and Diane Wright Center for Clinical and Translational Research, Virginia Commonwealth University, Richmond, VA 23298, United States.
Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA 23298, United States.
MethodsX. 2020 Nov 26;7:101163. doi: 10.1016/j.mex.2020.101163. eCollection 2020.
Extraction of high-quality RNA from pancreatic tumors for sequencing purposes is technically challenging, as the pancreas is an organ rich in ribonucleases. The majority of the established RNA isolation protocols for use with primary pancreatic tissue involve perfusion of RNA stabilizing reagent into the pancreatic tissue to protect RNA integrity before extraction. However, the additional time needed for this procedure can actually lead to further RNA degradation. We optimized a protocol suitable for high quality RNA isolation from mouse pancreatic tumors that is a simple, fast, and inexpensive modification of existing methods, combining the use of liquid nitrogen and guanidinium thiocyanate-chloroform extraction. Through this procedure, the mean RNA Integrity Number value obtained for RNA isolated from pancreatic tumors was 9.0, and was reproducibly suitable for RNAseq and qPCR.•a protocol suitable for high quality RNA isolation from mouse pancreatic tumors as well as normal pancreas•combining the use of liquid nitrogen and guanidinium thiocyanate-chloroform extraction.
从胰腺肿瘤中提取高质量RNA用于测序在技术上具有挑战性，因为胰腺是一个富含核糖核酸酶的器官。大多数已确立的用于原发性胰腺组织的RNA分离方案都涉及在提取前将RNA稳定试剂灌注到胰腺组织中以保护RNA完整性。然而，该过程所需的额外时间实际上可能导致进一步的RNA降解。我们优化了一种适用于从小鼠胰腺肿瘤中分离高质量RNA的方案，这是对现有方法的一种简单、快速且廉价的改进，结合了液氮和异硫氰酸胍 - 氯仿提取的使用。通过这个过程，从胰腺肿瘤中分离得到的RNA的平均RNA完整性数值为9.0，并且可重复地适用于RNA测序和定量聚合酶链反应。
•一种适用于从小鼠胰腺肿瘤以及正常胰腺中分离高质量RNA的方案
•结合使用液氮和异硫氰酸胍 - 氯仿提取
