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Assessment of RNA extraction protocols from cladocerans
Muhammad Raznisyafiq Razak1, Ahmad Zaharin Aris1,2, Fatimah Md Yusoff2,3
1Faculty of Forestry and Environment, Department of Environment, Universiti Putra Malaysia, Selangor, Malaysia.
Plos One
|April 26, 2022
Summary
This study found that a column-based kit is best for extracting RNA from Moina micrura, crucial for accurate gene expression analysis in ecotoxicology. Glycogen improved RNA recovery only with the phenol-chloroform method.
Area of Science:
- Environmental Science
- Molecular Biology
- Ecotoxicology
Background:
- Cladocerans are vital in aquatic ecosystems and increasingly used in ecotoxicological studies.
- Gene expression analysis in cladocerans requires high-quality extracted ribonucleic acid (RNA).
- Standardized RNA extraction methods for cladocerans are currently lacking.
Purpose of the Study:
- To evaluate two RNA extraction methods (phenol-chloroform vs. column-based kit) for tropical freshwater cladocerans (Moina micrura).
- To assess the impact of glycogen on RNA recovery in both methods.
- To validate the suitability of extracted RNA for downstream gene expression analysis using RT-PCR.
Main Methods:
- Extraction of RNA from Moina micrura using QIAzol (phenol-chloroform) and Qiagen Micro Kit (column-based).
- Inclusion of glycogen to enhance RNA yield in both methods.
- Characterization of RNA using NanoDrop (spectrophotometry) and Bioanalyzer (capillary electrophoresis).
- Validation of RNA quality and integrity via RT-PCR for alpha tubulin and actin gene amplification.
Main Results:
- The column-based kit yielded superior RNA quantity (26.90 ± 6.89 ng/μl), quality (A260:230 = 1.95 ± 0.15), and integrity (RIN = 7.20 ± 0.16).
- RT-PCR successfully amplified target genes (alpha tubulin, actin) from RNA extracted using the column-based kit (Ct = 32.64–33.48).
- Glycogen addition was beneficial only for the phenol-chloroform extraction method.
Conclusions:
- The column-based kit is the most suitable method for RNA extraction from Moina micrura for gene expression studies.
- Comprehensive quality control of extracted RNA is essential for reliable ecotoxicological data.
- This validated method can advance molecular-level ecotoxicological assessments in freshwater zooplankton.
