Optimizing Serum RNA Isolation: A Comparative Analysis of Commercial Kits for Yield, Purity, and Contamination Control
Abstract
Objective: Isolation of RNA from serum samples has gained importance, especially in studies on the use of small RNA molecules such as miRNA as biomarkers. Selection of the optimal kit is critical for the accuracy of downstream processing. The aim of this study was to compare the performance of different commercial kits in terms of efficiency, RNA purity and contamination control during the isolation process.
Methods: Three different RNA isolation kits were used for 5 serum samples: 1.miRNeasy Serum/Plasma Kit (Cat. No: 217184, Qiagen, USA), 2.Norgen Plasma/serum RNA purification kit (Cat. No: 55000, Norgen, Canada), 3.Nucleogene RNA isolation kit (Cat. No: NG044, Nucleogene, Turkey). The purity and intensity of the obtained RNAs were evaluated by measuring A260/280 ratios with a nanodrop spectrophotometer.
Results: When the concentrations and A260/280 ratios obtained from the kits were evaluated by One Way Anova Test using GraphPad Prism (V10.4.0), it was observed that there was a statistically significant difference between the concentrations and A260/280 ratios of the 3 kits (p≤0.05 and p≤ 0.001).
RNAs obtained from Norgene had the lowest concentration and the lowest A260/280 ratio, while Nucleogene had the highest RNA concentration and A260/280 ratio of 2 and above among the three kits (p≤0.05).
Ethical Statement
Approval was obtained before starting the study (2012/06/65)
Supporting Institution
No financial support was used in the study.
References
- Tanriverdi K, Kucukural A, Mikhalev E, et al. Comparison of RNA isolation and associated methods for extracellular RNA detection by high throughput quantitative polymerase chain reaction.Anal Biochem. 2016;501:66-74. doi:10.1016/j.ab.2016.02.019
- Tan SC, Yiap BC. DNA, RNA, and protein extraction: the past and the present [published correction appears in J Biomed Biotechnol. 2013;2013:628968]. J BiomedBiotechnol. 2009;2009:574398. doi:10.1155/2009/574398
- Wong RKY, MacMahon M, Woodside JV, Simpson DA. A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma. BMC Genomics. 2019;20(1):446. doi:10.1186/s12864-019-5826-7
- Roest HP, IJzermans JNM, van der Laan LJW. Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids. BMC Biotechnol. 2021;21(1):48. doi:10.1186/s12896-021-00706-6
- Sriram H, Khanka T, Kedia S, et al. Improved protocol for plasma microRNA extraction and comparison of commercial kits. BiochemMed (Zagreb). 2021;31(3):030705. doi:10.11613/BM.2021.030705
- Li Y, Kowdley KV. Method for microRNA isolation from clinical serum samples. Anal Biochem. 2012;431(1):69-75. doi:10.1016/j.ab.2012.09.007
- Chamberlain F, Grammatopoulos D. Methodology for Isolation of miRNA From the Serum of Women Investigated for Pre-eclampsia. Cureus. 2023;15(9):e46181. doi:10.7759/cureus.46181
- Khoury S, Ajuyah P, Tran N. Isolation of small noncoding RNAs from human serum. J Vis Exp. 2014;(88):e51443. doi:10.3791/51443
- Wilfinger WW, Eghbalnia HR, Mackey K, Miller R, Chomczynski P. Whole blood RNA extraction efficiency contributes to variability in RNA sequencing data sets. PLoS One. 2023;18(11):e0291209. doi:10.1371/journal.pone.0291209
- Brunet-Vega A, Pericay C, Quílez ME, Ramírez-Lázaro MJ, Calvet X, Lario S. Variability in microRNA recovery from plasma: Comparison of five commercial kits. Anal Biochem. 2015;488:28-35. doi:10.1016/j.ab.2015.07.018
- Chu CP, Nabity MB. Comparison of RNA isolation and library preparation methods for small RNA sequencing of canine biofluids. Vet Clin Pathol. 2019;48(2):310-319. doi:10.1111/vcp.12743
- Gautam A, Kumar R, Dimitrov G, Hoke A, Hammamieh R, Jett M. Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods. Mol Biol Rep. 2016;43(10):1165-1178. doi:10.1007/s11033-016-4043-6
Serum RNA İzolasyonunun Optimize Edilmesi: Verim, Saflık ve Kontaminasyon Kontrolü için Ticari Kitlerin Karşılaştırmalı Analizi
Abstract
Amaç: Serum örneklerinden RNA izolasyonu, özellikle miRNA gibi küçük RNA moleküllerinin biyo-belirteç olarak kullanımına yönelik çalışmalarda önem kazanmıştır. Optimal kitin seçimi sonraki işlemlerin doğruluğu için kritik öneme sahiptir. Bu çalışmanın amacı izolasyon sürecinde farklı ticari kitlerin verimlilik, RNA saflığı ve kontaminasyon kontrolü açısından performanslarını kıyaslamaktır.
Yöntem: Çalışmada 5 serum örneği için üç farklı RNA izolasyon kiti kullanılmıştır: 1. miRNeasy Serum/Plazma Kiti (Kat. No: 217184, Qiagen, ABD), 2. Norgen Plazma/serum RNA saflaştırma kiti (Kat. No: 55000, Norgen, Kanada), 3. Nucleogene RNA izolasyon kiti (Kat. No: NG044, Nucleogene, Türkiye). Elde edilen RNA’ların saflık ve yoğunluğu nanodrop spektrofotometre ile A260/280 oranları ölçülerek değerlendirilmiştir.
Bulgular: Kitlerden elde edilen konsantrasyonlar ve A260/280 oranları GraphPad Prism (V10.4.0) kullanılarak One Way Anova Testi ile değerlendirildiğinde, 3 kitin konsantrasyonları ve A260/280 oranları arasında istatistiksel olarak anlamlı bir fark olduğu görülmüştür (p≤0,05 ve p≤ 0,001).
Norgene'den elde edilen RNA'lar en düşük konsantrasyona ve en düşük A260/280 oranına sahipken, Nucleogene üç kit arasında en yüksek RNA konsantrasyonuna ve 2 ve üzeri A260/280 oranına sahipti (p≤0,05).
Sonuç: Serum RNA izolasyonu için kullanılan kitler arasında Nucleogene kiti genel olarak en yüksek RNA verimi ve uygun A260/280 değerleriyle öne çıkmaktadır. Bununla birlikte Qiagene ve Norgene kitleri bazı spesifik durumlarda tercih edilebilir.
References
- Tanriverdi K, Kucukural A, Mikhalev E, et al. Comparison of RNA isolation and associated methods for extracellular RNA detection by high throughput quantitative polymerase chain reaction.Anal Biochem. 2016;501:66-74. doi:10.1016/j.ab.2016.02.019
- Tan SC, Yiap BC. DNA, RNA, and protein extraction: the past and the present [published correction appears in J Biomed Biotechnol. 2013;2013:628968]. J BiomedBiotechnol. 2009;2009:574398. doi:10.1155/2009/574398
- Wong RKY, MacMahon M, Woodside JV, Simpson DA. A comparison of RNA extraction and sequencing protocols for detection of small RNAs in plasma. BMC Genomics. 2019;20(1):446. doi:10.1186/s12864-019-5826-7
- Roest HP, IJzermans JNM, van der Laan LJW. Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids. BMC Biotechnol. 2021;21(1):48. doi:10.1186/s12896-021-00706-6
- Sriram H, Khanka T, Kedia S, et al. Improved protocol for plasma microRNA extraction and comparison of commercial kits. BiochemMed (Zagreb). 2021;31(3):030705. doi:10.11613/BM.2021.030705
- Li Y, Kowdley KV. Method for microRNA isolation from clinical serum samples. Anal Biochem. 2012;431(1):69-75. doi:10.1016/j.ab.2012.09.007
- Chamberlain F, Grammatopoulos D. Methodology for Isolation of miRNA From the Serum of Women Investigated for Pre-eclampsia. Cureus. 2023;15(9):e46181. doi:10.7759/cureus.46181
- Khoury S, Ajuyah P, Tran N. Isolation of small noncoding RNAs from human serum. J Vis Exp. 2014;(88):e51443. doi:10.3791/51443
- Wilfinger WW, Eghbalnia HR, Mackey K, Miller R, Chomczynski P. Whole blood RNA extraction efficiency contributes to variability in RNA sequencing data sets. PLoS One. 2023;18(11):e0291209. doi:10.1371/journal.pone.0291209
- Brunet-Vega A, Pericay C, Quílez ME, Ramírez-Lázaro MJ, Calvet X, Lario S. Variability in microRNA recovery from plasma: Comparison of five commercial kits. Anal Biochem. 2015;488:28-35. doi:10.1016/j.ab.2015.07.018
- Chu CP, Nabity MB. Comparison of RNA isolation and library preparation methods for small RNA sequencing of canine biofluids. Vet Clin Pathol. 2019;48(2):310-319. doi:10.1111/vcp.12743
- Gautam A, Kumar R, Dimitrov G, Hoke A, Hammamieh R, Jett M. Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods. Mol Biol Rep. 2016;43(10):1165-1178. doi:10.1007/s11033-016-4043-6
Details
| Primary Language | English |
|---|---|
| Subjects | Bioinformatics and Computational Biology (Other) |
| Journal Section | Research Articles |
| Authors | |
| Publication Date | June 30, 2025 |
| Submission Date | January 28, 2025 |
| Acceptance Date | May 15, 2025 |
| Published in Issue | Year 2025 Volume: 8 Issue: 2 |
The articles in the Journal of "Acta Medica Nicomedia" are open access articles licensed under a Creative Commons Attribution-ShareAlike 4.0 International License at the web address https://dergipark.org.tr/tr/pub/actamednicomedia