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Letters to the Editor |
Departments of1 Chemical Pathology and2 Obstetrics and Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China
aAddress correspondence to this author at: Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, 30-32 Ngan Shing St., Shatin, New Territories, Hong Kong Special Administrative Region, China. Fax 852-2194-6171; e-mail loym{at}cuhk.edu.hk.
To the Editor:
Recent studies have shown that detection of fetal DNA or RNA in maternal plasma is useful for prenatal investigation of certain fetal genetic traits (1)(2) or pregnancy-associated complications (3)(4). Fetal DNA has been shown to amount to 3.4%–6.2% of the total DNA in maternal plasma (5). Thus, the reliability of circulating fetal nucleic acids analysis is dependent on the ability to sensitively and specifically detect and distinguish such fetal molecules from a background of maternal nucleic acids. Hence, methods that enable enrichment of the proportion of fetal nucleic acids in maternal plasma would, in theory, facilitate robust analysis of circulating fetal nucleic acids. Dhallan et al. (6) recently explored the use of formaldehyde for the enrichment of circulating fetal DNA. The authors reported marked increases in the proportion of fetal DNA in maternal blood samples preserved with formaldehyde. Although controversies exist regarding the effect of formaldehyde on fetal DNA enrichment (7)(8), we nonetheless wanted to investigate whether formaldehyde could be used for the enrichment of circulating fetal RNA.
We collected EDTA-blood samples (12 mL) from each of 6 pregnant women (gestational age, 17–19 weeks) with informed consent. We immediately treated 6 mL of each blood sample with 150 µL of 10% neutral-buffered solution containing formaldehyde and left the remaining 6 mL untreated; we then subdivided the treated and untreated blood portions into 3 aliquots. One aliquot each from the treated and untreated groups was processed by centrifugation to obtain plasma (9), either immediately or after 6 or 24 h of storage at 4 °C. RNA was extracted from the plasma by a combined Trizol LS and column-based method as described previously (9) and was further subjected to real-time quantitative reverse transcription-PCR analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (9) (Fig. 1A
) and a placenta-specific mRNA, human placental lactogen (hPL) mRNA (10) (data not shown), were detected in the maternal plasma aliquots without formaldehyde treatment but in none of the paired aliquots treated with formaldehyde.
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Because formaldehyde has previously been shown to interact with Trizol and affect its ability to extract RNA from tissue (11), we investigated whether this was the cause of our observed data. Thus, instead of adding formaldehyde directly to the whole-blood samples, we mixed formaldehyde with Trizol LS and used that solution to extract RNA from plasma samples that had not been treated previously with formaldehyde. RNA was readily detectable from plasma samples extracted with Trizol LS solutions containing up to 2 mL/L formaldehyde, which was equivalent to the final concentration of formaldehyde in the blood samples evaluated in the earlier part of the study, but not when the formaldehyde concentration in the solution was 20 mL/L (Fig. 1B
). These data suggest that formaldehyde does affect the efficacy of RNA extraction by Trizol LS, but not at the concentration used to preserve the maternal blood samples. We then used a non-Trizol–based extraction method, the QIAamp viral RNA Mini Reagent Kit (Qiagen), to extract plasma RNA from blood samples to which formaldehyde had or had not been added. RNA was not amplifiable from any of the plasma extracts obtained from the formaldehyde-treated blood (Fig. 1C
).
Although the underlying mechanism is unclear, based on these results, we conclude that formaldehyde has a detrimental effect on plasma RNA detection. Irrespective of the extraction protocol used, it appears that no amplifiable RNA in plasma can be obtained from formaldehyde-treated whole-blood samples.
References
The following articles in journals at HighWire Press have cited this article:
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  E C W Hung, R W K Chiu, and Y M D Lo Detection of circulating fetal nucleic acids: a review of methods and applications J. Clin. Pathol., April 1, 2009; 62(4): 308 - 313. [Abstract] [Full Text] [PDF]
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 Y. M. Dennis Lo and R. W. K. Chiu Noninvasive Prenatal Diagnosis of Fetal Chromosomal Aneuploidies by Maternal Plasma Nucleic Acid Analysis Clin. Chem., March 1, 2008; 54(3): 461 - 466. [Abstract] [Full Text] [PDF]
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