Modified Trizol Method for Extracting RNA from Mouse Skin / 中山大学学报(医学科学版)

ABSTRACT

@#【Objective】Due to the tough nature of skin tissue and a high presence of RNases，the isolation of skin RNA by the classical Trizol method presents a challenge. Therefore，we adapted and tested different sample treatment protocols to improve the Trizol method for high- quality extraction of skin RNA.【Methods】In this study，normal skin of mice processed by different treatments（Tri：submersion Trizol；Pro：RNA sample protector；Cry：cryopreservation in liquid nitrogen frozen and then - 80 ℃ refrigerator；LNG：liquid nitrogen grinding；Cut：scissor cutting）were used as the experimental groups. Spinal cord tissue（Sp）was used as the reference group，and skin tissue of mouse psoriasis model induced by imiquimod（IMQ）was used as the validation group. We compared skin RNA concentration，purity and integrity， as well as IL- 1β mRNA expression extracted by conventional Trizol methods（1-Tri，Nor）and modified Trizol methods（2-Tri，LNG-Tri，Tri-Cut，Pro），which were determined by UV spectrophotometry，agar gel electrophoresis and quantitative reverse transcription PCR（qRT- PCR）.【Results】① Compared with spinal cord（Sp），the total RNA of normal skin tissue extracted with the same classical Trizol method（1-Tri）was with lower yields，more obvious DNA contamination and 5S RNA bands，and higher IL-1β mRNA relative expression，suggesting that skin tissue was relatively special and the classical Trizol methods of skin RNA extraction should be improved. ② Among the different treatment methods of skin tissue，2-Tri and LNG-Tri methods resulted in higher RNA concentrations，lower RNA degradation and lower DNA contamination，and the expression of IL-1β mRNA was closer to normal levels. More importantly，the skin RNA samples extracted by the 2-Tri method can reflect more realistically the variation of IL-1β mRNA expression between normal and psoriasiform groups.【Conclusion】Improved 2-Tri or LNG-Tri method has the advantage of high quality of total RNA，and 2-Tri can more reliably reflect the mRNA expression pattern under physiological and pathological conditions.