Scholarlink

Background <p>RNA biomarker research has grown significantly in recent years, with a focus on blood-based biomarker studies using cell-free nucleic acids (cf-NAs). These RNA derivatives (e.g., mRNAs, microRNAs, and long non-coding RNAs) are useful in diagnostic procedures for genetic changes and can be found in a variety of physiological fluids, such as plasma, brain fluid, and urine. RNA biomarkers are composed of different coding and non-coding transcripts. Among these, cfRNAs extracted from blood provide a minimally invasive source for disease diagnosis and monitoring. However, cfRNA isolation is challenging due to degradation, instability, lack of standardization, and contamination by microbial, environmental, and intrasample DNA. Therefore, it is crucial to obtain cell-free RNAs in high concentration using appropriate methods and process them using downstream PCR systems.</p> Methods and results <p>To compare the purity and quality of cfRNAs isolated from plasma, five different isolation methods using commercial cfRNA kits were evaluated by digital PCR (dPCR) and quantitative PCR (qPCR). RNA quality was assessed across kits, and expression levels were measured using cfRNA reference genes such as <i>GAPDH</i> and <i>B2M</i>. The results of analysis revealed that the <i>GAPDH</i> housekeeping gene showed greater consistency than <i>B2M</i>, and both genes exhibited statistically significant expression.</p> Conclusions <p>Our comparative analysis of five cfRNA isolation methods demonstrated that commercial kits outperformed phenol–chloroform–based procedures. These results underscore the need for optimized isolation strategies and careful reference gene selection to enhance the reliability of cfRNA-based biomarker discovery.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Evaluation of Plasma-Derived Cell-Free RNA isolation methods using PCR-Based quantification

  • 期刊论文

  • 发表日期：

    2026年1月3日

查看全文
  • Gizem Inetas-Yengin,
  • Zehra Omeroglu-Ulu,
  • Fikrettin Sahin,
  • Dilek Telci

摘要

Background

RNA biomarker research has grown significantly in recent years, with a focus on blood-based biomarker studies using cell-free nucleic acids (cf-NAs). These RNA derivatives (e.g., mRNAs, microRNAs, and long non-coding RNAs) are useful in diagnostic procedures for genetic changes and can be found in a variety of physiological fluids, such as plasma, brain fluid, and urine. RNA biomarkers are composed of different coding and non-coding transcripts. Among these, cfRNAs extracted from blood provide a minimally invasive source for disease diagnosis and monitoring. However, cfRNA isolation is challenging due to degradation, instability, lack of standardization, and contamination by microbial, environmental, and intrasample DNA. Therefore, it is crucial to obtain cell-free RNAs in high concentration using appropriate methods and process them using downstream PCR systems.

Methods and results

To compare the purity and quality of cfRNAs isolated from plasma, five different isolation methods using commercial cfRNA kits were evaluated by digital PCR (dPCR) and quantitative PCR (qPCR). RNA quality was assessed across kits, and expression levels were measured using cfRNA reference genes such as GAPDH and B2M. The results of analysis revealed that the GAPDH housekeeping gene showed greater consistency than B2M, and both genes exhibited statistically significant expression.

Conclusions

Our comparative analysis of five cfRNA isolation methods demonstrated that commercial kits outperformed phenol–chloroform–based procedures. These results underscore the need for optimized isolation strategies and careful reference gene selection to enhance the reliability of cfRNA-based biomarker discovery.