Newborns are routinely screened for a variety of conditions—predominantly inborn errors of metabolism—for which early diagnosis can lead to therapy and subsequent prevention of sequelae, including permanent neurological damage [1
]. Such screening programs are in place throughout the world. In the United States, mandatory screening programs exist in all 50 states; the number of conditions screened for in each state varies from 4 to 30 [2
]. Within 24-48 hours after birth, newborn blood is collected on filter paper cards (“Guthrie cards”) and submitted for laboratory analysis using principally tandem mass spectrometry, high performance liquid chromatography, and antibody based assays. These assays generally consume only a small portion of the available blood on the cards. The portion of the cards with remaining blood is then archived under varying conditions, and for varying durations. A recent survey found that 14 of the 51 US newborn screening programs retain newborn blood for 21 years or more [3
]. These population-wide archives of biologic specimens represent a unique resource for the retrospective study of diseases of perinatal origin.
To be of use in health research, informative material in dried blood spots must persist after years of dry, room temperature storage. Proteins in blood spots are beyond detection in a matter of days [4
], but nucleic acids show evidence of long-term stability. The published literature shows extensive examples of recovery and analysis of nucleic acids from dried blood spots. Many studies have involved screening for viral and bacterial DNA and RNA in dried blood spots [5
]. The presence of infectious agents at birth may be very relevant to understanding diseases of perinatal origin, but since analysis of nucleic acids from infectious agents is relatively well documented we focus our attention on DNA and RNA from the neonate.
Most reports on the analysis of neonatal nucleic acids from archived blood spots have focused on DNA, and several procedures for extracting DNA from filter paper have been published [10
]. Subsequent analyses of blood spot DNA have included on-chip PCR based microarray screening for specific gene polymorphisms, [14
], screening for disease markers and mutations [15
] and post-mortem diagnosis of a disease-causing mutation [19
]. Another interesting use of archived neonatal blood DNA has been to use PCR and sequencing to identify leukemic cells present at birth and other diagnostic markers in infants who subsequently developed leukemia [20
As RNA is known to be more labile than DNA, it is reasonable to doubt the stability of RNA in long term unfrozen storage. Yet peer-reviewed research shows that RNA in dried blood spots is stable after years of storage, contains transcripts indicative of specific diseases, and mirrors the gene expression in fresh blood. Blood spot RNA analysis was first published by Matsabura [24
] and Zhang [23
], who performed mutation and restriction enzyme analysis of expressed genes from spots stored for several years. Long-term RNA stability has been most strongly demonstrated by Swedish researchers who have recovered intact RNA transcripts from blood spots archived up to 21 years. [25
]. Several researchers show that genes expressed in archived blood samples reflect disease states. Suttorp detected RNA for the bcr-abl rearrangement indicative of the Philadelphia chromosome in dried blood spots [26
]. Pai showed that patients who lack expression of the FMR-1 gene due to fragile-X syndrome lack FMR-1 RNA in their neonatal blood spot [27
]. Both Pai and Suttorp verified their blood spot findings against RNA from fresh blood. We found one paper that compared RNA from a sample of fresh blood against RNA from identical blood spotted, dried, and stored for several months [28
]. Maeno confirmed that, after several months of room temperature storage, infection-induced cytokine mRNA in the blood spot precisely mirrored the genes expressed in the original fresh blood sample. While these publications all support the stability of blood spot RNA and its abilility to reflect disease states, they only examined from 1 to 13 RNA transcripts at a time.
Researchers have yet to apply the latest tools in RNA technology to measure blood spot gene expression on a larger scale. We can find no published reports of microarray-based gene expression analysis of RNA from archived neonatal blood spots. If whole-expressed genome quantitative RNA expression can be applied to archived newborn blood spots, these specimens would constitute a remarkable resource for clinicians and researchers. Moreso than DNA, blood spot RNA would provide a temporal molecular snapshot of physiologic and metabolic function shortly after birth. These “expressed genetic profiles” could then be retrospectively studied to identify markers and potential causal factors of childhood (and perhaps adult-onset) diseases from the perinatal period. RNA data could add further value to the already universally collected (and in some states research-available) neonatal blood spot.
In this report, we describe the results of parallel microarray-based gene expression and quantitative PCR analyses of RNA extracted from 12 archived neonatal blood spots.