Carrier RNA (cRNA) Enhances dsDNA Recovery Extracted from Small Volume Spent Embryo Culture Medium

Ayu Mulia Sundari, Pritta Ameilia Iffanolida, Kresna Mutia, Naylah Muna, Eliza Mansyur, Andon Hestiantoro, Budi Wiweko, Muhammad Fauzi, Anom Bowolaksono


Embryo spent culture medium has been intensively investigated, considering its promising feature for non-invasive bioanalytical techniques in in-vitro fertilization (IVF). Despite, isolating DNA from such samples is quite challenging due to its small volume. Carrier RNA is reported to exhibit DNA retrieval effects and commonly employed in various limited biological samples, but there are no reports regarding its benefit on embryo media. Therefore, we aim to evaluate the competence of cRNA on isolated DNA from embryo medium and analyzed its optimal volume as there are also no records respecting its ideal volume to obtain decent outcomes. Results showed that cRNA significantly increases DNA amounts in the cRNA treated group (p<0.001), but the D-4/D-5 medium yielded similar (p=0.684). Pearson test demonstrated no correlation between cRNA volume vs. total retrieved DNA (r=0.760, p=0.80), and Whole Genome Amplification (WGA) was shown to increase DNA in the treated group (p=0.022), but not in the untreated group (p=0.128). Additionally, electrophoresis successfully resulting in a thick and thin band of TH01 locus signifies the cRNA competence. In conclusion, our study suggests that cRNA addition is essential in embryo medium extraction as it increases initial DNA that crucial for downstream application. However, the optimal volume could not be determined in the current study since the initial amount of DNA in the medium is unknown. Obtained findings are expected to be a new input for subsequent research on DNA extraction.


Assisted reproductive technology; carrier RNA; in-vitro fertilization; spent embryo medium.

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M. R. Sadeghi, “The 40th anniversary of IVF: Has ART’s success reached its peak?,†J. Reprod. Fertil., vol. 19, no. 2. Avicenna Research Institute, pp. 67–68, Apr. 01, 2018, Accessed: Feb. 17, 2021. [Online]. Available:

B. Wiweko et al., “Correlation between luteinizing hormone receptor gene expression in human granulosa cells with oocyte quality in poor responder patients undergoing in vitro fertilization: A cross-sectional study [version 1; peer review: 2 approved, 1 approved with reserv,†F1000Res, vol. 8, no. 16, pp. 1–13, 2019, doi: 10.12688/f1000research.17036.1.

S. Moustafa and S. Young, “Diagnostic and therapeutic options in recurrent implantation failure,†F1000Res, vol. 9, no. 1, pp. 1–9, 2020, doi: 10.12688/f1000research.22403.1.

J. Patel, A. Patel, J. Banker, S. Shah, and M. Banker, “Personalized embryo transfer helps in improving In vitro fertilization/ICSI outcomes in patients with recurrent implantation failure,†J. Hum. Reprod. Sci., vol. 12, no. 1, pp. 59–66, Jan. 2019, doi: 10.4103/jhrs.JHRS_74_18.

M. ReljiÄ, J. Knez, V. KovaÄ, and B. KovaÄiÄ, “Endometrial injury, the quality of embryos, and blastocyst transfer are the most important prognostic factors for in vitro fertilization success after previous repeated unsuccessful attempts,†J. Assist. Reprod. Genet., vol. 34, no. 6, pp. 775–779, Jun. 2017, doi: 10.1007/s10815-017-0916-4.

F. Fesahat, F. Montazeri, M. H. Sheikhha, H. Saeedi, R. D. Firouzabadi, and S. M. Kalantar, “Frequency of chromosomal aneuploidy in high quality embryos from young couples using preimplantation genetic screening,†Int J Reprod Biomed, vol. 15, no. 5, pp. 297–304, May 2017, doi: 10.29252/ijrm.15.5.297.

R. Sciorio and M. Dattilo, “PGT-A preimplantation genetic testing for aneuploidies and embryo selection in routine ART cycles: Time to step back?,†Clin. Genet, vol. 98, no. 2, pp. 107–115, Aug. 2020, doi: 10.1111/cge.13732.

X. W. Gu et al., “ATP mediates the interaction between human blastocyst and endometrium,†Cell Prolif., vol. 53, no. 2, p. e12737, Feb. 2020, doi: 10.1111/cpr.12737.

L. Guzman et al., “The number of biopsied trophectoderm cells may affect pregnancy outcomes,†J. Assist. Reprod. Genet., vol. 36, no. 1, pp. 145–151, Jan. 2019, doi: 10.1007/s10815-018-1331-1.

L. Huang, B. Bogale, Y. Tang, S. Lu, X. S. Xie, and C. Racowsky, “Noninvasive preimplantation genetic testing for aneuploidy in spent medium may be more reliable than trophectoderm biopsy,†in Proc. Natl. Acad. Sci. U. S. A., 2019, vol. 116, no. 28, pp. 14105–14112, doi: 10.1073/pnas.1907472116.

V. Kuznyetsov et al., “Minimally Invasive Cell-Free Human Embryo Aneuploidy Testing (miPGT-A) Utilizing Combined Spent Embryo Culture Medium and Blastocoel Fluid –Towards Development of a Clinical Assay,†Sci. Rep., vol. 10, no. 1, p. 7244, Dec. 2020, doi: 10.1038/s41598-020-64335-3.

A. V. Timofeeva, V. V. Chagovets, Y. S. Drapkina, N. P. Makarova, E. A. Kalinina, and G. T. Sukhikh, “Cell-free, embryo-specific sncRNA as a molecular biological bridge between patient fertility and IVF efficiency,†Int. J. Mol. Sci., vol. 20, no. 12, p. 2912, Jun. 2019, doi: 10.3390/ijms20122912.

L. Yang et al., “Presence of embryonic DNA in culture medium,†Oncotarget, vol. 8, no. 40, pp. 67805–67809, Sep. 2017, doi: 10.18632/oncotarget.18852.

S. Stigliani et al., “Non-invasive mitochondrial DNA quantification on Day 3 predicts blastocyst development: A prospective, blinded, multi-centric study,†Mol. Hum. Reprod., vol. 25, no. 9, pp. 527–537, Aug. 2019, doi: 10.1093/molehr/gaz032.

D. Cimadomo et al., “Definition and validation of a custom protocol to detect miRNAs in the spent media after blastocyst culture: Searching for biomarkers of implantation,†Hum. Reprod., vol. 34, no. 9, pp. 1746–1761, Sep. 2019, doi: 10.1093/humrep/dez119.

F. Carvalho et al., “ESHRE PGT Consortium good practice recommendations for the detection of monogenic disorders†,†Hum. Reprod. Open, vol. 2020, no. 3, pp. 1–18, Mar. 2020, doi: 10.1093/hropen/hoaa018.

C. Katevatis, A. Fan, and C. M. Klapperich, “Low concentration DNA extraction and recovery using a silica solid phase,†PLoS One, vol. 12, no. 5, p. e0176848, May 2017, doi: 10.1371/journal.pone.0176848.

D. Higgins, J. Kaidonis, G. Townsend, and J. J. Austin, “Evaluation of carrier RNA and low volume demineralization for recovery of nuclear DNA from human teeth,†Forensic Sci Med Pathol, vol. 10, no. 1, pp. 56–61, Mar. 2014, doi: 10.1007/s12024-013-9519-2.

K. J. Shaw et al., “The use of carrier RNA to enhance DNA extraction from microfluidic-based silica monoliths,†Anal. Chim. Acta, vol. 652, no. 1–2, pp. 231–233, 2009, doi: 10.1016/j.aca.2009.03.038.

S. I. Pearlman et al., “Low-Resource Nucleic Acid Extraction Method Enabled by High-Gradient Magnetic Separation,†ACS Appl. Mater. Interfaces, vol. 12, no. 11, pp. 12457–12467, Mar. 2020, doi: 10.1021/acsami.9b21564.

R. Kishore, W. Reef Hardy, V. J. Anderson, N. A. Sanchez, and M. R. Buoncristiani, “Optimization of DNA extraction from low-yield and degraded samples using the BioRobot® EZ1 and BioRobot® M48,†J. Forensic Sci., vol. 51, no. 5, pp. 1055–1061, Sep. 2006, doi: 10.1111/j.1556-4029.2006.00204.x.

L. Galluzzi et al., “Extracellular embryo genomic DNA and its potential for genotyping applications,†Futur. Sci. OA, vol. 1, no. 4, p. FSO62, Nov. 2015, doi: 10.4155/fso.15.62.

P. A. Reddy, M. Bhavanishankar, J. Bhagavatula, K. Harika, R. S. Mahla, and S. Shivaji, “Improved methods of carnivore faecal sample preservation, DNA extraction and quantification for accurate genotyping of wild tigers,†PLoS One, vol. 7, no. 10, p. e46732, Oct. 2012, doi: 10.1371/journal.pone.0046732.

M. Vera-Rodriguez et al., “Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development,†Hum. Reprod., vol. 33, no. 4, pp. 745–756, Apr. 2018, doi: 10.1093/humrep/dey028.

S. Hughes, N. Arneson, S. Done, and J. Squire, “The use of whole genome amplification in the study of human disease,†Prog. Biophys. Mol., vol. 88, no. 1. Prog Biophys Mol Biol, pp. 173–189, May 2005, doi: 10.1016/j.pbiomolbio.2004.01.007.

H. Wahyuningsih et al., “Optimization of PCR condition: The first study of high-resolution melting technique for screening of APOA1 variance,†Yonago acta med, vol. 60, no. 1, pp. 24–30, Mar. 2017, Accessed: Feb. 17, 2021. [Online]. Available:



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