One-phase phenol-free method for microRNA isolation from blood plasma

Area of Science:

• Molecular Biology
• Biochemistry
• Biomarker Discovery

Background:

• MicroRNA extraction is crucial for identifying microRNA-based biomarkers and therapeutic strategies.
• Existing methods for microRNA isolation from human blood plasma can be cumbersome or inefficient.

Purpose of the Study:

• To develop and validate a novel, efficient method for microRNA isolation from human blood plasma.
• To optimize microRNA precipitation from a one-phase lysate using isopropanol.

Main Methods:

• A new microRNA isolation protocol based on isopropanol precipitation from a one-phase lysate was developed.
• Optimization involved varying lysis buffer volumes, using linear polyacrylamide (LPAA) with co-precipitants (poly(A)RNA or tRNA), and substituting dithiothreitol for β-mercaptoethanol.
• The novel method was compared against the miRNeasy Mini Kit (Qiagen) for microRNA yield from human blood plasma.

Main Results:

• Using over four volumes of 5 M guanidine isothiocyanate lysis buffer prevented precipitate issues.
• Linear polyacrylamide alone was ineffective, but adding poly(A)RNA or tRNA significantly boosted microRNA yield.
• The new method demonstrated superior microRNA recovery, evidenced by lower median Ct values for both exogenous cel-238 (Δ median Ct 4.94) and endogenous microRNA-21 (Δ median Ct 2.18) compared to the Qiagen kit.

Conclusions:

• The described isopropanol precipitation method offers a high-yield and convenient alternative for microRNA isolation from human blood plasma.
• This method requires no specialized equipment, making it accessible for various research settings.
• The improved recovery facilitates more effective microRNA biomarker discovery and analysis.