TY - JOUR

T1 - How Does the Sample Preparation of Phytophthora infestans Mycelium Affect the Quality of Isolated RNA?

AU - Ivanov, Artemii A

AU - Tyapkin, Alexandr V

AU - Golubeva, Tatiana S

N1 - Funding: This research was supported by a grant from the President of the Russian Federation for state support of young Russian scientists (MK-4311.2022.5).

PY - 2023/4/18

Y1 - 2023/4/18

N2 - RNA isolation from fungi and fungus-like organisms is not an easy task. Active endogenous RNases quickly hydrolyze RNA after the sample collection, and the thick cell wall prevents inhibitors from penetrating the cells. Therefore, the initial collection and grinding steps may be crucial for the total RNA isolation from the mycelium. When isolating RNA from Phytophthora infestans, we varied the grinding time of the Tissue Lyser and used TRIzol and beta-mercaptoethanol to inhibit the RNase. In addition, we tested the mortar and pestle grinding of mycelium in liquid nitrogen, with this method showing the most consistent results. During the sample grinding with the Tissue Lyser device, adding an RNase inhibitor proved to be a prerequisite, and the best results were achieved using TRIzol. We considered ten different combinations of grinding conditions and isolation methods. The classical combination of a mortar and pestle, followed by TRIzol, has proved to be the most efficient.

AB - RNA isolation from fungi and fungus-like organisms is not an easy task. Active endogenous RNases quickly hydrolyze RNA after the sample collection, and the thick cell wall prevents inhibitors from penetrating the cells. Therefore, the initial collection and grinding steps may be crucial for the total RNA isolation from the mycelium. When isolating RNA from Phytophthora infestans, we varied the grinding time of the Tissue Lyser and used TRIzol and beta-mercaptoethanol to inhibit the RNase. In addition, we tested the mortar and pestle grinding of mycelium in liquid nitrogen, with this method showing the most consistent results. During the sample grinding with the Tissue Lyser device, adding an RNase inhibitor proved to be a prerequisite, and the best results were achieved using TRIzol. We considered ten different combinations of grinding conditions and isolation methods. The classical combination of a mortar and pestle, followed by TRIzol, has proved to be the most efficient.

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85153726676&origin=inward&txGid=8d3b01841f3146821538b1f2112819b5

U2 - 10.3390/cimb45040230

DO - 10.3390/cimb45040230

M3 - Article

C2 - 37185754

VL - 45

SP - 3517

EP - 3524

JO - Current issues in molecular biology

JF - Current issues in molecular biology

SN - 1467-3037

IS - 4

ER -