Long-term conservation of HCV RNA at 4 degrees C using a new RNA stabilizing solution.
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.
Gonzalez-Perez
Idania
I
Centro de InmunoEnsayo (CIE), Cubanacán, Playa, Apartado Postal, CP 11600 Ciudad de la Habana, Cuba. idania_gonzalez@yahoo.es
Cayarga
Anny Armas
AA
Hernández
Yenitse Perea
YP
de la Rosa
Iria García
IG
González
Yaimé Josefina González
YJ
León
Carlos Silva
CS
Alvarez
René Robaina
RR
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*
D002021:Buffers; D004791:Enzyme Inhibitors; D012367:RNA, Viral; D013861:Thiocyanates; C026976:thiocyanic acid; D019791:Guanidine