title: Blood cell mRNAs and microRNAs: optimized protocols for extraction and preservation.
abstract: Assessing messenger RNA (mRNA) and microRNA levels in peripheral blood cells may complement conventional parameters in clinical practice. Working with small, precious samples requires optimal RNA yields and minimal RNA degradation. Several procedures for RNA extraction and complementary DNA (cDNA) synthesis were compared for their efficiency. The effect on RNA quality of freeze-thawing peripheral blood cells and storage in preserving reagents was investigated. In terms of RNA yield and convenience, quality quantitative polymerase chain reaction signals per nanogram of total RNA and using NucleoSpin and mirVana columns is preferable. The SuperScript III protocol results in the highest cDNA yields. During conventional procedures of storing peripheral blood cells at -180°C and thawing them thereafter, RNA integrity is maintained. TRIzol preserves RNA in cells stored at -20°C. Detection of mRNA levels significantly decreases in degraded RNA samples, whereas microRNA molecules remain relatively stable. When standardized to reference targets, mRNA transcripts and microRNAs can be reliably quantified in moderately degraded (quality index 4-7) and severely degraded (quality index <4) RNA samples, respectively. We describe a strategy for obtaining high-quality and quantity RNA from fresh and stored cells from blood. The results serve as a guideline for sensitive mRNA and microRNA expression assessment in clinical material.
authors: Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands. m.eikmans@lumc.nl
authors: Michael
authors: M
authors: Eikmans
authors: Niels V
authors: NV
authors: Rekers
authors: Jacqueline D H
authors: JD
authors: Anholts
authors: Sebastiaan
authors: S
authors: Heidt
authors: Frans H J
authors: FH
authors: Claas
mesh_terms: D000465:Algorithms; D001773:Blood Cells / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism*; D001774:Blood Chemical Analysis / Q000379:methods / Q000592:standards; D001793:Blood Preservation / Q000379:methods / Q000592:standards; D001800:Blood Specimen Collection / Q000379:methods* / Q000592:standards; D002138:Calibration; D005821:Genetic Techniques / Q000592:standards; D006146:Guanidines; D006801:Humans; D035683:MicroRNAs / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D010636:Phenols; D016133:Polymerase Chain Reaction / Q000379:methods; D011309:Preservation, Biological / Q000379:methods* / Q000592:standards; D020871:RNA Stability / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D012015:Reference Standards
chemical_list: D006146:Guanidines; D035683:MicroRNAs; D010636:Phenols; D012333:RNA, Messenger; C411644:trizol