BACKGROUND: Purification of mRNA from stored specimens is very important because results from RT-PCR and microarray analyses are largely affected by the quality of mRNA. Moreover, many preanalytical factors during collection, processing, and storage may affect mRNA quality and the expression of peripheral blood mononuclear cells (PBMC). In this study, we evaluate the effects of RBC removal techniques and TRIzol on RNA quality in blood samples. METHODS: We obtained EDTA-blood samples from 50 adult volunteers, and made 10 pools of buffy coats for comparison between protocols and also evaluated RNA quality of clinical samples in biobank. Use of TRIzol and RBC removal (RBC lysis or cell separation) were evaluated their effect on the quality of mRNA from the stored blood samples. RESULTS: RNA integrity with TRIzol was significantly better than that without TRIzol (RIN 4.5 vs. 9.2, respectively; P=0.002). The change in RIN of the PBMC separation method was equivalent to that of the RBC lysis method. After 12 months, IL6 mRNA expression from stored clinical samples in cell separation/TRIzol was stable. CONCLUSIONS: The blood samples frozen in TRIzol after RBC removal preserved RNA quality well. PBMC/TRIzol preservation for storage of blood samples could be a simple protocol for rapid, low-cost biobanking.
BACKGROUND: Purification of mRNA from stored specimens is very important because results from RT-PCR and microarray analyses are largely affected by the quality of mRNA. Moreover, many preanalytical factors during collection, processing, and storage may affect mRNA quality and the expression of peripheral blood mononuclear cells (PBMC). In this study, we evaluate the effects of RBC removal techniques and TRIzol on RNA quality in blood samples. METHODS: We obtained EDTA-blood samples from 50 adult volunteers, and made 10 pools of buffy coats for comparison between protocols and also evaluated RNA quality of clinical samples in biobank. Use of TRIzol and RBC removal (RBC lysis or cell separation) were evaluated their effect on the quality of mRNA from the stored blood samples. RESULTS: RNA integrity with TRIzol was significantly better than that without TRIzol (RIN 4.5 vs. 9.2, respectively; P=0.002). The change in RIN of the PBMC separation method was equivalent to that of the RBC lysis method. After 12 months, IL6 mRNA expression from stored clinical samples in cell separation/TRIzol was stable. CONCLUSIONS: The blood samples frozen in TRIzol after RBC removal preserved RNA quality well. PBMC/TRIzol preservation for storage of blood samples could be a simple protocol for rapid, low-cost biobanking.
Authors: Duncan E Donohue; Aarti Gautam; Stacy-Ann Miller; Seshamalini Srinivasan; Duna Abu-Amara; Ross Campbell; Charles R Marmar; Rasha Hammamieh; Marti Jett Journal: PLoS One Date: 2019-10-10 Impact factor: 3.240
Authors: Christopher P Corkum; Danielle P Ings; Christopher Burgess; Sylwia Karwowska; Werner Kroll; Tomasz I Michalak Journal: BMC Immunol Date: 2015-08-26 Impact factor: 3.615