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

Abstract

Background: 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. Methods: Filter papers (ns5) 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 b-actin. Results: Transcripts encoding b-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. Conclusions: 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.

Real-Time Quantitative Reverse Transcription PCR for Monitoring of Blood-Stage Plasmodium falciparum Infections in Malaria Human Challenge Trials

“…To reap the benefits of the high copy number 18S rRNA as a target, samples must be adequately preserved at the time of collection, which is easily accomplished using guanidiniumbased lysis buffer and, possibly, even dried blood spots. [49][50][51] Given the need for reliable, controlled assays to support malaria clinical trials and eradication efforts, we recommend the use of multiplexed internal control targets and in vitro transcribed RNA standards for absolute quantification in all RNA-or total nucleic acid-based assays. Furthermore, a network for inter-laboratory exchange of well-characterized malaria comparator samples would be useful to help laboratories establish and maintain assay performance.…”

Current status of newborn screening worldwide: 2015

“…82,83 Researchers have examined various specimen stability issues. 84,85 There has also been considerable discussion about privacy and consent. 86,87 While there is experimentation and discussion aimed at expanding current NBS panels to include one or more LSDs, expansion is unlikely to happen on a large scale.…”

Newborn Screening By Tandem Mass Spectrometry: Impacts, Implications and Perspectives

“…On the other hand, DNA quality from stored DBS specimens at room temperature allowed extraction and successful amplification for at least 25 years (Searles Nielsen et al, 2008). Quantitative RNA stability was also shown from stored residual NBS specimens for 20 years at 4 0 C in controlled relative humidity maintained at 30% (Gauffin et al, 2009). Specimen-to-specimen contamination should be prevented.…”