Detectability of circulating microRNAs in microRNA extracts with low purity and yield using quantitative real-time polymerase chain reaction: Supporting evidence

Authors

Azmir Ahmad, Department of Basic Medical Science for Nursing, Kulliyyah of Nursing, International Islamic University Malaysia, Kuantan 25200, MalaysiaFollow
Mohd. Arifin Kaderi, Department of Biomedical Science, Kulliyyah of Allied Health Sciences, International Islamic University Malaysia, Kuantan 25200, MalaysiaFollow
Afidalina Tumian, PETRONAS Research Sdn. Bhd., Bandar Baru Bangi 43000, MalaysiaFollow
Vijaya Mohan Sivanesan, Molecular Pathology Unit, Cancer Research Centre, Institute for Medical Research, Shah Alam 40170, MalaysiaFollow
Kahairi Abdullah, KPJ Batu Pahat Specialist Hospital, Batu Pahat 83000, MalaysiaFollow
Wan Ishlah Leman, Department of Otorhinolaryngology-Head & Neck Surgery, Kulliyyah of Medicine, International Islamic University Malaysia, Kuantan 25200, MalaysiaFollow
Irfan Mohamad, Department of Otorhinolaryngology-Head & Neck Surgery, School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu 16150, MalaysiaFollow
Wan Mohd. Nazri Wan Zainon, Department of Nuclear Medicine, Radiotherapy and Oncology, School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu 16150, MalaysiaFollow
Muhammad Izani Mohd. Shiyuti, Department of Otorhinolaryngology, Hospital Tengku Ampuan Afzan, Kuantan 25100, MalaysiaFollow
Kamariah Mohamed Awang, Department of Otorhinolaryngology, Hospital Tengku Ampuan Afzan, Kuantan 25100, Malaysia
Luqman Rosla, Department of Otorhinolaryngology, Hospital Sultan Haji Ahmad Shah, Temerloh 28000, MalaysiaFollow
Mark Paul, Department of Otorhinolaryngology, Hospital Sultan Haji Ahmad Shah, Temerloh 28000, MalaysiaFollow
Sharifah Nor Ezura Syed Yussof, Department of Otorhinolaryngology, Hospital Sultan Haji Ahmad Shah, Temerloh 28000, Malaysia
Rosdi Ramli, Department of Otorhinolaryngology, Hospital Raja Perempuan Zainab II, Kota Bharu 15200, Malaysia

Abstract

Background: Circulating microRNAs (miRNAs) are a group of noncoding RNAs with promising potential as minimal invasive biomarkers for noncommunicable diseases. However, challenges exist in the preparation of these miRNAs from peripheral blood samples for quantification purposes. The low quality of miRNA extracts presents an obstacle. Acknowledging the superior performance of quantitative real-time polymerase chain reaction (qPCR) as gold standard for gene expression analysis, we conducted this study to observe the capabilities of qPCR using the Taqman® protocol in amplifying circulating miRNAs from miRNA extracts with low purity and yield.

Methods: miRNAs were extracted from thirty-six plasma samples that were obtained from public subjects. Four selected miRNAs were quantified using the Taqman® protocol in an integrated fluidic circuit chip that was optimized from a previous study. The amplification graph and Cq values were obtained to observe any abnormal amplification signs and expression levels, respectively.

Results: The qualitative observation of the amplification of the four miRNAs showed no sign of abnormality, thereby indicating the successful amplification of the miRNAs without enzymatic inhibition. Furthermore, the miRNAs were quantified in high expression levels.

Conclusion: The circulating miRNA extracts with low purity and yield were practical for the study of circulating miRNA expression based on the Taqman® protocol as the method of detection.

References

1. Rolle K, Piwecka M, Belter A, Wawrzyniak D, Jeleniewicz J, Barciszewska MZ, et al. The sequence and structure determine the function of mature human miRNAs. PLoS One. 2016;11:e0151246.

2. O’Brien J, Hayder H, Zayed Y, Peng C. Overview of microRNA biogenesis, mechanism of actions, and circulation. Front Endocrinol (Lausanne). 2018;9:402.

3. Hanna J, Hossain GS, Kocerha J. The potential of microRNA therapeutics and clinical research. Front Genet. 2019;10:478.

4. Michlewski G, Cáceres JF. Post-transcriptional control of miRNA biogenesis. RNA. 2019;25:1–16.

5. Turunen TA, Roberts TC, Laitinen P, Väänänen MA, Korhonen P, Malm T, et al. Changes in nuclear and cytoplasmic microRNA distribution in response to hypoxic stress. Sci Rep. 2019;9:10332.

6. Di Mauro V, Crasto S, Colombo FS, Di Pasquale E, Catalucci D. Wnt signalling mediates miR-133a nuclear re-localization for the transcriptional control of Dnmt3b in cardiac cells. Sci Rep. 2019;9:9320.

7. Jin Y, Wong YS, Goh BKP, Chan CY, Cheow PC, Chow PKH, et al. Circulating microRNAs as potential diagnostic and prognostic biomarkers in hepatocellular carcinoma. Sci Rep. 2019;9:10464.

8. Wu L, Zheng K, Yan C, Pan X, Liu Y, Liu J, et al. Genome-wide study of salivary microRNAs as potential noninvasive biomarkers for detection of nasopharyngeal carcinoma. BMC Cancer. 2019;19:843.

9. Park Y. MicroRNA exocytosis by vesicle fusion in neuro-endocrine cells. Front Endocrinol (Lausanne). 2017;8:355.

10. Sohel MH. Extracellular/circulating microRNAs: Release mechanisms, functions and challenges. Achiev Life Sci. 2016;10:175–86.

11. Guo Y, Vickers K, Xiong Y, Zhao S, Sheng Q, Zhang P, et al. Comprehensive evaluation of extracellular small RNA isolation methods from serum in high throughput sequencing. BMC Genomics. 2017;18:50.

12. Max KEA, Bertram K, Akat KM, Bogardus KA, Li J, Morozov P, et al. Human plasma and serum extracellular small RNA reference profiles and their clinical utility. Proc Natl Acad Sci USA. 2018;115:E5334–43.

13. Poel D, Buffart TE, Oosterling-Jansen J, Verheul HMW, Voortman J. Evaluation of several methodological challenges in circulating miRNA qPCR studies in patients with head and neck cancer. Exp Mol Med. 2018;50:e454.

14. Myklebust MP, Rosenlund R, Gjengstø P, Bercea BS, Karlsdottir Á, Brydøy M, et al. Quantitative PCR measurement of miR-371a-3p and miR-372-p is influenced by haemolysis. Front Genet. 2019;10:463.

15. Babafemi EO, Cherian BP, Banting L, Mills GA, Ngianga K.2nd. Effectiveness of real-time polymerase chain reaction assay for the detection of Mycobacterium tuberculosis in pathological samples: A systematic review and meta-analysis. Syst Rev. 2017;6:215.

16. Barbieri RR, Manta FSN, Moreira SJM, Sales AM, Nery JAC, Nascimneto LPR, et al. Quantitative polymerase chain reaction in paucibacillary leprosy diagnosis: A follow-up study. PLoS Negl Trop Dis. 2019;13:e0007147.

17. Miranda JA, Steward GF. Variables influencing the efficiency and interpretation of reverse transcription quantitative PCR (RT-qPCR): An empirical study using bacteriophage MS2. J Virol Methods. 2017;241:1–10.

18. Ruiz-Villalba A, van Pelt-Verkuil E, Gunst QD, Ruijter JM, van den Hoff MJ. Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR). Biomol Detect Quantif. 2017;14:7–18.

19. Moret I, Sánchez-Izquierdo D, Iborra M, Tortosa L, Navarro-Puche A, Nos P, et al. Assessing an improved protocol for plasma microRNA extraction. PLoS One. 2013;8:e82753.

20. Wang B, Howel P, Bruheim S, Ju J, Owen LB, Fodstad O, et al. Systematic evaluation of three microRNA profiling platforms: Microarray, beads array and quantitative real-time PCR array. PLoS One. 2011;6:e17167.

21. Crane SL, van Dorst J, Hose GC, King CK, Ferrari BC. Microfluidic qPCR enables high throughput quantification of microbial functional genes but requires strict curation of primers. Front Environ Sci. 2018;6:145.

22. Tuck MK, Chan DW, Chia D, Godwin AK, Grizzle WE, Krueger KE, et al. Standard operating procedures for serum and plasma collection: Early detection research network consensus statement standard operating procedure integration working group. J Proteome Res. 2009;8:113–7.

23. Wozniak MB, Scelo G, Muller DC, Mukeria A, Zaridze D, Brennan P. Circulating microRNAs as non-invasive biomarkers for early detection for non-small-cell lung cancer. PLoS One. 2015;10:e0125026.

24. Tiberio P, Callari M, Angeloni V, Daidone MG, Appierto V. Challenges in using circulating miRNAs as cancer biomarkers. BioMed Res Int 2015;2015:731479.

25. El-Khoury V, Pierson S, Kaoma T, Bernardin F, Berchem G. Assessing cellular and circulating miRNA recovery: The impact of the RNA isolation method and the quantity of input material. Sci Rep. 2016;6:19529.

26. Gao X, Xie Z, Wang Z, Cheng K, Liang K, Song Z. overexpression of miR-191 predicts poor prognosis and promotes proliferation and invasion in esophageal squamous cell carcinoma. Yonsei Med J. 2017;58:1101–10.

27. Akhbari M, Shahrabi-Farahani M, Biglari A, Khalili M, Bandarian F. Expression level of circulating miR-93 in serum of patients with diabetic nephropathy. Turk J Endocrinol Metab. 2018;22:78–84.

28. Alharthi A, Beck D, Howard DR, Hillmen P, Oates M, Pettitt A, et al. An increased fraction of circulating miR-363 and miR-16 is particle bound in patients with chronic lymphocytic leukaemia as compared to normal subjects. BMC Res Notes. 2018;11:280.

29. Shafiei J, Javadi G, Nateghi B, Shaygannejad V, Salehi M. Up-regulation of circulating miR-93-5p in patients with relapsing-remitting multiple sclerosis. J Bas Res Med Sci. 2019;6:4–11.

30. Zbucka-Kretowska M, Niemira M, Paczkowska-Abdulsalam M, Bielska A, Szalkowska A, Parfieniuk E, et al. Prenatal circulating microRNA signatures of foetal Down syndrome. Sci Rep. 2019;9:2394.

31. Reis PP, Drigo SA, Carvalho RF, Lapa RML, Felix TF, Patel D, et al. Circulating miR-16-5p, miR-92a-3p, and miR-451a in plasma from lung cancer patients: Potential application in early detection and a regulatory role in tumorigenesis pathway. Cancers. 2020;12:2017.

32. Taylor SC, Nadeau K, Abbasi M, Lachance C, Nguyen M, Fenrich J. The ultimate qPCR experiment: Producing publication quality, reproducible data the first time. Trends Biotechnol. 2019;37:761–74.

33. Desjardins P, Conklin D. NanoDrop microvolume quantitation of nucleic acids. J Vis Exp. 2010;45:2565.

34. Hedman J, Rådström P. Overcoming inhibition in real-time diagnostic PCR. Methods Mol Biol. 2013;943:17–48.

35. Sidstedt M, Romsos EL, Hedell R, Ansell R, Steffen CR, Vallone PM, et al. Accurate digital polymerase chain reaction quantification of challenging samples applying inhibitor-tolerant DNA polymerases. Anal Chem. 2017;89:1642–49.

36. Sidstedt M, Hedman J, Romsos EL, Waitara L, Wadsö L, Steffen CR, et al. Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR. Anal Bioanal Chem. 2018;410:2569–83.

37. Schwochow D, Serieys LEK, Wayne RK, Thalmann O. Efficient recovery of whole blood RNA - A comparison of commercial RNA extraction protocols for high-throughput applications in wildlife species. BMC Biotechnol. 2012;12:33.

38. Rossen L, Nørskov P, Holmstrøm K, Rasmussen OF. Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int J Food Microbiol. 1992;17:37–45.

39. Lucena-Aguilar G, Sánchez-López AM, Barberán-Aceituno C, Carrillo-Ávila JA, López-Guerrero JA, Aguilar-Quesada R. DNA source selection for downstream applications based on DNA quality indicators analysis. Biopreserv Biobank. 2016;14:264–70.

40. Correia CN, McLoughlin KE, Nalpas NC, Magee DA, Browne JA, Rue-Albrecht K, et al. RNA sequencing (RNA-Seq) reveals extremely low levels of reticulocyte-derived globin gene transcripts in peripheral blood from horses (Equus caballus) and cattle (Bos taurus). Front Genet. 2018;9:278.

41. Mumford SL, Towler BP, Pashler AL, Gilleard O, Martin Y, Newbury SF. Circulating microRNA biomarkers in melanoma: Tools and challenges in personalised medicine. Biomolecules. 2018;8:E21.

42. Syed Ahmad Kabeer B, Tomei S, Mattei V, Brummaier T, McGready R, Nosten F, et al. A protocol for extraction of total RNA from finger stick whole blood samples preserved with Tempus™ solution [version 1; peer review: 2 approved with reservations]. F1000Research. 2018;7:1739.

43. Marabita F, de Candia P, Torri A, Tegnér J, Abrignani S, Rossi RL. Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR. Brief Bioinform. 2016;17:204–12.

44. Khetan D, Gupta N, Chaudhary R, Shukla JS. Comparison of UV spectrometry and fluorometry-based methods for quantification of cell-free DNA in red cell components. Asian J Transfus Sci. 2019;13:95–9.

45. Tan GW, Khoo ASB, Tan LP. Evaluation of extraction kits and RT-qPCR system adapted to high-throughput platform for circulating miRNAs. Sci Rep. 2015;5:9430.

46. Vigneron N, Meryet-Figuière M, Guttin A, Issartel JP, Lambert B, Briand M, et al. Towards a new standardized method for circulating miRNAs profiling in clinical studies: Interest of the exogenous normalization to improve miRNA signature accuracy. Mol Oncol. 2016;10:981–92.

47. Burdukiewicz M, Spiess AN, Blagodatskikh KA, Lehmann W, Schierack P, Rödiger S. Algorithms for automated detection of hook effect-bearing amplification curves. Biomol Detect Quantif. 2018;16:1–4.

48. Jansson L, Hedman J. Challenging the proposed causes of the PCR plateau phase. Biomol Detect Quantif. 2019;17:100082.

49. Wang J, Yu JT, Tan L, Tian Y, Ma J, Tan CC, et al. Genome-wide circulating microRNA expression profiling indicates biomarkers for epilepsy. Sci Rep. 2015;5:9522.

50. Kramer MF. Stem-loop RT-qPCR for miRNAs. Curr Protoc Mol Biol. 2011;Chapter 15:Unit 15.10.

51. Le Carré J, Lamon S, Léger, B.Validation of a multiplex reverse transcription and pre-amplification method using Taqman® microRNA assays. Front Genet. 2014;5:413.

52. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 2005;33:e179.

53. Jung U, Jiang X, Kaufmann SHE, Patzel V. A universal Taqman-based RT-PCR protocol for cost-efficient detection of small noncoding RNA. RNA. 2013;19:1864–73.

54. Chen Y, Gelfond JA, McManus LM, Shireman PK. Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis. BMC Genomics. 2009;10:407.

55. Korenková V, Scott J, Novosadová V, Jindřichová M, Langerová L, Švec D, et al. Pre-amplification in the context of high-throughput qPCR gene expression experiment. BMC Mol Biol. 2015;16:5.

56. Venkatesan G, Bhanuprakash V, Balamurugan V, Prabhu M, Pandey AB. Taqman hydrolysis probe based real time PCR for detection and quantitation of camelpox virus in skin scabs. J Virol Methods. 2012;181:192–6.

57. Tajadini M, Panjehpour M, Javanmard SH. Comparison of SYBR Green and Taqman methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes. Adv Biomed Res. 2014;3:85.

58. El-Sayed AKA, Abou-Dobara MI, Abdel-Malak CA, El-Badaly AAE. Taqman hydrolysis probe application for Escherichia coli, Salmonella enterica, and Vibrio cholerae detection in surface and drinking water. J Water Sanit Hyg Dev. 2019;9:492–9.

59. Wang T, Brown MJ. mRNA quantification by real time Taqman polymerase chain reaction: Validation and comparison with RNase protection. Anal Biochem. 1999;269:198–201.

60. VanGuilder HD, Vrana KE, Freeman WM. Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques. 2008;44:619–26.

Recommended Citation

Ahmad A, Kaderi M, Tumian A, Sivanesan VM, Abdullah K, Leman W, et al. Detectability of circulating microRNAs in microRNA extracts with low purity and yield using quantitative real-time polymerase chain reaction: Supporting evidence. Makara J Health Res. 2020;24.