Quantitation of RNA decay in dried blood spots during 20 years of storage

Abstract

Diseases with an onset during childhood or adult life can have their origin during fetal life or at birth. Neonatal blood dried on filter paper (Guthrie cards) collected for screening purposes is routinely stored for decades. In addition to clinical use, these filters in combination with patient registers constitute an invaluable resource for epidemiological and pathophysiological research. Although RNA has been successfully recovered from such filters even after decades of storage, the potential decay of RNA over time has not previously been investigated using quantitative methods. Filter papers (n=5) with dried blood spots from the Swedish National PKU register, stored for 1, 5, 10, 15 or 20 years were randomly selected. RNA was isolated from each sample, quantitated by spectrophotometry and reverse transcribed following DNase I treatment. Amplifiable cDNA was subsequently detected by real-time PCR using primers specific for transcripts encoding beta-actin. Transcripts encoding beta-actin were detected in all 25 samples analyzed at a mean threshold cycle (Ct) of 25 (SD 1.9). A one-way ANOVA indicated no significant effect of storage time on Ct values. The lack of significant decay of RNA in dried blood filters stored for up to 20 years suggests that such filters are useful for studies of RNA determinants of diseases with an onset in childhood as well as adult life.