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Nutrition and Health (including climate and ecological aspects)

Placental expression of RNU44, RNU48 and miR-16-5p: stability and relations with fetoplacental growth

Abstract

Background/Objectives

The current study aimed to identify suitable reference miRNA for placental miRNA expression analysis in a set of well-characterized and fetal-sex balanced small- (SGA) and appropriate- (AGA) for gestational age full-term singleton pregnancies.

Subjects/Methods

In this retrospective study, placental samples (n = 106) from 35 SGA (19 male and 16 female) and 71 AGA (30 male and 41 female) full-term singleton pregnancies were utilized. Placental transcript abundance of three widely used reference miRNAs [miR-16-5p and Small nucleolar RNAs (snoRNAs) RNU44 and RNU48] were assessed by real-time quantitative PCR. Raw cycle threshold (Ct) analysis and RefFinder tool analysis were conducted for evaluating stability of expression of these miRNAs.

Results

Raw Ct values of miR-16-5p were similar between SGA and AGA births (P = 0.140) and between male and female births within SGA (P = 0.159) and AGA (P = 0.060) births while that of RNU44 and RNU48 were higher in SGA births (P = 0.008 and 0.006 respectively) and in male births within the SGA group (P = 0.005) for RNU44 and in female births within the AGA group (P = 0.048) for RNU48. Across all 106 samples tested using the RefFinder tool, miR-16-5p and RNU44 were equally stable reference miRNAs.

Conclusion

We recommend miR-16-5p and RNU44 as suitable reference miRNAs for placental samples from settings similar to our study.

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Fig. 1: Distribution of RNU44, RNU48 and miR-16-5p expression [cycle threshold (Ct) values] in placental tissue from 35 SGA and 71AGA births.
Fig. 2: Stability of the reference genes (RNU44, RNU48 and miR-16-5p) using multiple algorithms.

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Acknowledgements

We thank the pregnant women who participated in the study and doctors and nurses who made this study possible. The contribution of the research assistants Ms. Nancy N, Ms. Roopashree C, Ms. Aruna BS and Ms. Arogya M who collected the samples and data and of histopath technician Ms. Mahalakshmi S assisted the pathologists in placental grossing experiments is acknowledged. This work was supported by the Women Scientist Scheme, Department of Science & Technology, Government of India to PK (Reference no. SR/WOS-A/LS-669/2016) and the Department of Biotechnology, Government of India grants to AM and AVK (Grant sanction nos. BT/PR22326/MED/97/349/2016 and BT/PR30276/MED/97/399/2018).

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AM, AVK and PK designed the study. AM and PK contributed to the planning of the experiments. PD, GR, AT and JC provided samples and clinical information for the study. PK performed all experiments. AM and TT conducted the statistical analyses. AM and PK wrote the manuscript. AM, AVK, TT and PD contributed to the interpretation of the results and manuscript writing. All authors discussed the results, have seen and approved the final version of the manuscript.

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Correspondence to A. Mukhopadhyay.

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Kochhar, P., Dwarkanath, P., Ravikumar, G. et al. Placental expression of RNU44, RNU48 and miR-16-5p: stability and relations with fetoplacental growth. Eur J Clin Nutr (2021). https://doi.org/10.1038/s41430-021-01003-3

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