Measuring fetal brain and lung transcripts in amniotic fluid supernatant: a comparison of digital PCR and RT-qPCR methods

Abstract

Purpose: Amniotic fluid (AF) cell-free RNA is a promising source of information regarding fetal physiology. Digital PCR (dPCR) is a direct approach to nucleic acid detection that reports absolute transcript copy number. The aim of this study was to compare quantification of cell-free fetal brain and lung RNA transcripts in AF by reverse transcription-qPCR (RT-qPCR) and dPCR.

Material and methods: Prospective hospital-based study was performed in 2016–2017. Pulmonary genes were quantified in term AF samples collected at elective cesarean birth; neurodevelopmental genes were measured in preterm samples (<34 weeks) obtained from women undergoing clinically-indicated amniocentesis.

Results: All 11 women in the term cohort had three lung transcripts and a reference gene successfully amplified from their AF supernatant using RT-qPCR and dPCR. SFTPC was the most abundant lung transcript, present in higher concentrations than the reference gene in seven of the eleven samples. Neurodevelopmental gene transcripts in 12 preterm pregnancies were less reliably detected by both methods and were present in low copy numbers (<10 copies/μl). We observed significant positive correlations between transcript quantification by RT-qPCR and dPCR.

Conclusion: This study confirms the presence of several potential mRNA markers of lung and brain development with dPCR and RT-qPCR, and a high correlation between the two methods. Transcripts of presumed fetal brain origin are present in very low copy numbers, which presents challenges to their feasibility as biomarkers of neurodevelopment.

Keywords:

• Amniotic fluid
• cell-free
• cell-free RNA
• digital PCR
• fetal brain
• fetal development
• gene expression
• lung maturity
• mRNA
• neurodevelopment
• RNA
• RT qPCR

Acknowledgements

The authors thank the research midwives and clinical staff of the Mercy Hospital for Women (Heidelberg, Victoria) for patient recruitment and sample collection of the term cohort, and the staff of the Victorian Clinical Genetics Service for sampling handling and storage of the preterm cohort.

Ethics and consent to participate

The Mercy Health Human Research Ethics Committee approved this study (Ref. no. R14/20). All participants gave written informed consent for participation in the study.

Health and safety

All mandatory laboratory health and safety procedures have been complied with in the course of conducting the experimental work reported in this paper.

Disclosure statement

The authors report no conflicts of interest.

Funding

Funding for this study was provided by the Norman Beischer Medical Research Foundation, the Sylvia Charles Viertel Charitable Foundation, and the Academic Research and Development Committee of Mercy Health Services. The National Health and Medical Research Council provided salary support to L. H. (# 1105603). N. J. H. is supported by a University of Melbourne CR Roper Fellowship. None of the funders had any involvement in the study design, data collection, data analysis, manuscript preparation or publication decision.