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Technical Briefs |
1 Division of Neurodegenerative Diseases, Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden
2 PKU Laboratory, Center for Inherited Metabolic Diseases, Huddinge University Hospital, 141 86 Huddinge, Sweden
aaddress correspondence to this author at: Department of Neuroscience, Karolinska Institutet, Retzius väg 8, S-171 77 Stockholm, Sweden; fax 46-8-32-53-25, e-mail hakan.karlsson{at}neuro.ki.se
Blood dried on filter paper is widely used for screening of inherited metabolic disorders (1). In Sweden, such filters from all newborns have been permanently stored since 1975. It has been shown that proteins and DNA may be recovered from these cards after extended periods of storage (2)(3)(4). RNA, however, has been considered too vulnerable to degradation by ribonucleases to be recovered from these filters. Despite this, Zhang and McCabe (5) and Matsubara et al.(6) reported that mRNA could be isolated from such filters after up to 4 years of storage. The stability of viral RNA on filters has also been reported (7)(8), although this was not tested over extended periods of time. The purpose of the present study was to investigate whether RNA could be recovered from filters that had been stored since 1975 and be amplified by reverse transcription-PCR.
After approval by the local ethics committee, we randomly selected filter papers (specimen collection paper 2992; Schleicher & Schuell) that had been stored for 1 month, 21 years, and 27 years; for each time point, we selected five filters. One-fourth of a spot (
0.3 cm2) containing dried blood was cut out of each filter with a sterile razor blade. As a negative control, a piece of corresponding size was cut from a blood-free area of each filter. RNA was isolated from the specimen with use of the RNeasy reagent set (Qiagen) according to the manufacturer’s instructions. Briefly, filters were incubated in lysis buffer at 37 °C for 30 min in a thermomixer (Eppendorf) rotating at 1000 rpm. The lysates were subsequently homogenized by spinning through a QiaShredder (Qiagen). The flow-through was applied to a RNeasy column, and after careful washing, the RNA was eluted in 50 µL of RNase-free water. We subjected 8 µL of the RNA to DNase I digestion (Life Technologies) with subsequent reverse transcription in a 20-µL reaction with the following reagents from Life Technologies: 150 ng of random primers, 1x first strand buffer, 10 mM dithiothreitol, and 0.5 mM each of the deoxynucleotide triphosphates. The reaction was heated to 72 °C and chilled on ice before the addition of 200 U of Superscript II. cDNA was then generated for 1 h at 42 °C before the reaction was inactivated by heating to 70 °C for 15 min.
The presence of cDNA was verified by PCR using primers designed to amplify fragments of the mRNAs encoding human ß-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; see Table 1
), respectively, using the following conditions: 1 µL of cDNA was added to a 24-µL reaction mixture containing 1x Titanium Taq DNA Polymerase mixture; 1x Titanium PCR Buffer (Clontech Laboratories); 1 µM each of gene-specific forward and reverse primers, respectively; and 200 µM each of the deoxynucleotide triphosphates (Life Technologies). For amplification, a GeneAmp PCR system 9700 (Applied Biosystems) was used at the following cycling conditions: heat activation for 2 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 30s and annealing/extension at 68 °C for 60 s, with a final extension at 72 °C for 7 min. PCR products were electrophoresed in 2% agarose in Tris-acetate-EDTA buffer (40 mmol/L Tris, 20 mmol/L acetic acid, 1 mmol/L EDTA). Double-stranded DNA was stained in 1x SYBR Gold Stain (Molecular Probes) in the Tris-acetate-EDTA buffer. Double-stranded DNA was subsequently visualized and documented on a Gel Doc 2000 system (Bio-Rad).
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We were able to amplify 97- and 205-bp mRNA fragments encoding human GAPDH and ß-actin, respectively, from filters that had been stored for 1 month, 21 years, and 27 years (Fig. 1A
). No RNA was detected on the filters outside the area of the dried blood spot. Because GAPDH- and ß-actin-related pseudogenes are present in the human genome, generation of an intronless amplicon does not rule out the presence of contaminating genomic DNA in the RNA preparation. DNase I treatment of the RNA before the generation of cDNA is crucial, and documented that RNA was present (Fig. 1B
).
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Although filters have been stored since 1975, only since 1981 have all filters been kept at 4 °C. Additionally, only since 1996 have all filters been stored in an area with a controlled relative humidity not exceeding 30%. This difference in storage conditions did not seem to greatly influence the amount or quality of the RNA that was amplified. The total amount of recovered RNA was insufficient for meaningful spectrophotometric quantification, and no efforts were made to quantify the amount of starting material by the PCR approach used.
These filters constitute a unique resource for investigating events that may be reflected in peripheral blood cells or plasma around the time of birth, and they have previously been used for DNA and protein analysis. Our present finding expands on the number of investigations that can be performed with these filters. One such application may be testing hypotheses put forward by epidemiologists regarding various disorders of an autoimmune or developmental nature. For several of these, exposure to infectious agents during the intrauterine period or early life has been suggested as a risk factor for developing the disease (9)(10).
In conclusion, routinely stored phenylketonuria-screening filters may be used for the study of perinatal events, detectable in RNA, that may be of relevance for the etiopathogenesis of disorders that would be very costly and, today, impractical to address in prospective studies.
Acknowledgments
This study was supported by The Stanley Medical Research Institute.
References
The following articles in journals at HighWire Press have cited this article:
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  U Hannelius, C M Lindgren, E Melen, A Malmberg, U von Dobeln, and J Kere Phenylketonuria screening registry as a resource for population genetic studies J. Med. Genet., October 1, 2005; 42(10): e60 - e60. [Abstract] [Full Text] [PDF]
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