title: Long-term conservation of HCV RNA at 4 degrees C using a new RNA stabilizing solution.
abstract: Protecting RNA from degradation, whilst maintaining its biological activity, is essential in molecular biology. However, RNA is very sensitive to degradation by ribonucleases, especially at temperatures above 0 degrees C. The stability of RNA was examined at 4 degrees C and -20 degrees C, in a new stabilizing solution consisting of a low-molarity mixture of chaotropic agents guanidinium and ammonium thiocyanate, a buffer for pH stabilization, phenol, and yeast RNA. Two substrates were tested for storage: RNA in human plasma positive for hepatitis C virus (HCV) and naked RNA (purified from HCV positive human plasma or transcribed in vitro). Stability was followed by viral load estimation, using an in-house competitive RT-PCR assay. Naked RNA purified from human plasma positive for HCV was stable at 4 degrees C for at least 24 months. An RNA standard transcribed in vitro was still viable after 36 months of storage at 4 degrees C. Human plasma dilutions positive for HCV were stable for at least 5 months in this solution when stored at 4 degrees C. It was concluded that the described stabilizing solution ensures long-term stability on naked RNA at 4 degrees C, and ideal for the storage of RNA controls and standards for molecular diagnosis, the solution may be used for preserving clinical samples prior to transport to a clinical laboratory.
authors.affiliation: Centro de InmunoEnsayo (CIE), Cubanacán, Playa, Apartado Postal, CP 11600 Ciudad de la Habana, Cuba. idania_gonzalez@yahoo.es
authors.forename: Idania
authors.initials: I
authors.lastname: Gonzalez-Perez
authors.forename: Anny Armas
authors.initials: AA
authors.lastname: Cayarga
authors.forename: Yenitse Perea
authors.initials: YP
authors.lastname: Hernández
authors.forename: Iria García
authors.initials: IG
authors.lastname: de la Rosa
authors.forename: Yaimé Josefina González
authors.initials: YJ
authors.lastname: González
authors.forename: Carlos Silva
authors.initials: CS
authors.lastname: León
authors.forename: René Robaina
authors.initials: RR
authors.lastname: Alvarez
mesh_terms: D002021:Buffers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019791:Guanidine / Q000494:pharmacology; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D012034:Refrigeration / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013048:Specimen Handling / Q000379:methods*; D013861:Thiocyanates / Q000494:pharmacology; D013997:Time Factors; D014773:Virology / Q000379:methods*
chemical_list: D002021:Buffers; D004791:Enzyme Inhibitors; D012367:RNA, Viral; D013861:Thiocyanates; C026976:thiocyanic acid; D019791:Guanidine