A microRNA isolation method from clinical samples.

ABSTRACT:

Introduction

microRNAs (miRNAs) are considered to be novel molecular biomakers that could be exploited in the diagnosis and treatment of different diseases. The present study aimed to develop an efficient miRNA isolation method from different clinical specimens.

Methods

Total RNAs were isolated by Trizol reagent followed by precipitation of the large RNAs with potassium acetate (KCH3COOH), polyethylene glycol (PEG) 4000 and 6000, and lithium chloride (LiCl). Then, small RNAs were enriched and recovered from the supernatants by applying a combination of LiCl and ethanol. The efficiency of the method was evaluated through the quality, quantity, and integrity of the recovered RNAs using the A260/280 absorbance ratio, reverse transcription PCR (RT-PCR), and quantitative real-time PCR (q-PCR).

Results

Comparison of different RNA isolation methods based on the precipitation of DNA and large RNAs, high miRNA recovery and PCR efficiency revealed that applying potassium acetate with final precipitation of small RNAs using 2.5 M LiCl plus ethanol can provide high yield and quality small RNAs that can be exploited for clinical purposes.

Conclusion

The current isolation method can be applied for most clinical samples including cells, formalin-fixed and paraffin-embedded (FFPE) tissues and even body fluids with a wide applicability in molecular biology investigations.

SUBMITTER: Zununi Vahed S 

PROVIDER: S-EPMC4916548 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature