MicroRNAs (miRNAs) are crucial in post-transcriptional regulation and are implicated in various diseases. Accurate miRNA profiling is vital for developing precision medicine, requiring high-quality sample preparation. Current miRNA extraction methods, often based on silica-based solid-phase extraction, can vary in efficiency. This research focuses on optimizing a commercial silica-based miRNA extraction kit by investigating the underlying mechanisms of miRNA adsorption and elution to enhance recovery from cultured cells. The purity and quality of extracted miRNAs are critical for reliable downstream analyses such as qPCR, microarray, and NGS, ensuring accurate diagnostic and prognostic biomarker assessments.

Nucleic acid extraction methods are broadly classified into liquid-phase and solid-phase extraction. Liquid-phase methods, such as phenol-chloroform extraction, utilize organic solvents to separate RNA. Solid-phase methods, prevalent in commercial kits, leverage silica-based sorbents. The adsorption of nucleic acids onto silica is influenced by factors such as chaotropic agents (like guanidinium), pH, ionic strength (monovalent and divalent cations), and temperature. Studies have shown that divalent cations (Ca²⁺, Mg²⁺) enhance RNA adsorption due to salt bridge formation between negatively charged silica and nucleic acid backbones. Ethanol or isopropanol reduce the polarity of the solution, facilitating adsorption. While many commercial kits use silica-based extraction, variations in recovery highlight the potential for optimization. Previous research has explored these factors for larger nucleic acids, but the optimal conditions for miRNA extraction remain less defined.

This study used HCT 116 human colon cancer cells. MiRNA extraction was performed using the miRNeasy Mini Kit (QIAGEN), with modifications to the adsorption and elution steps. Three ethanol concentrations (60%, 65%, 70%) were tested for adsorption, along with the addition of calcium chloride (5mM). For elution, the temperature (room temperature vs. 55°C) and pH (using RNase-free water vs. TE buffer, pH 8.0) were varied. Synthetic miR-39 was spiked-in to assess recovery. Endogenous miR-21 and U6 snRNA were quantified using qPCR. The qPCR utilized TaqMan MicroRNA Reverse Transcription Kit and TaqMan® Small RNA Assays (Life Technologies) with appropriate master mixes and cycling conditions. Statistical analyses (one-way ANOVA with post-hoc Tukey test) were performed using SPSS (version 18.0) to compare Ct values across different conditions. A calibration curve for miR-21 was generated using serially diluted synthetic miR-21 to determine the concentration of extracted miR-21.

The researchers observed significant improvements in miRNA recovery with modifications to both the adsorption and elution steps. In the elution process, raising the temperature to 55°C reduced the Ct values of both miR-21 and miR-39, suggesting increased recovery. This is consistent with the disruption of hydrogen bonds between the miRNA and silica. The use of TE buffer (pH 8.0) further enhanced elution efficiency, although the effect was more pronounced at the elevated temperature. The addition of calcium chloride (5 mM) during adsorption did not improve miRNA recovery. This might be because the high concentration of guanidine isothiocyanate (around 1M) in the adsorption buffer already efficiently shields the negative charges, and additional calcium ions may interfere with hydrogen bond formation, especially impacting smaller miRNAs. However, increasing the ethanol concentration during adsorption to 65% (v/v) significantly reduced Ct values for miR-21, demonstrating enhanced miRNA binding to the silica column. This is attributed to the decreased solubility of miRNA and increased dehydration effects. Combining the optimal conditions (65% ethanol for adsorption, TE buffer at 55°C for elution), the modified protocol yielded a sixfold increase in miR-21 recovery compared to the original method, as determined from the calibration curve. The Ct values of U6 (a reference snRNA) were similar between the original and modified methods, although there was a statistically significant difference.

This study successfully optimized the miRNA extraction protocol by modifying the adsorption and elution steps. The findings demonstrate that adjusting ethanol concentration and elution temperature and buffer significantly enhances miRNA recovery. The lack of improvement with Ca²⁺ addition highlights the complexity of the interaction between miRNAs, silica, and the existing chaotropic agent. The modified protocol improves the quality and quantity of miRNA extracted from cultured cells, ultimately improving the reliability and accuracy of downstream analyses. The results underscore the importance of optimizing extraction procedures tailored to the specific target molecule (miRNA) and the conditions of the extraction kit being used.

This research provides a significantly improved method for miRNA extraction from cultured cells using readily available reagents. The optimization of ethanol concentration during adsorption and the use of TE buffer with elevated temperature during elution yielded a sixfold increase in miR-21 recovery. This optimized protocol enhances the reliability of miRNA-based research and clinical diagnostics by providing higher yields of high-quality miRNA.

The study focused solely on HCT 116 cells. The generalizability of these findings to other cell types or tissues requires further investigation. While U6 snRNA served as a reference gene, potential variations in U6 expression under different experimental conditions should be considered. The modified method requires a 55°C incubation step during elution, which may not be feasible for all laboratory setups.