Assessment of RNA extraction protocols from cladocerans.

The usage of cladocerans as non-model organisms in ecotoxicological and risk assessment studies has intensified in recent years due to their ecological importance in aquatic ecosystems. The molecular assessment such as gene expression analysis has been introduced in ecotoxicological and risk assessment to link the expression of specific genes to a biological process in the cladocerans. The validity and accuracy of gene expression analysis depends on the quantity, quality and integrity of extracted ribonucleic acid (RNA) of the sample. However, the standard methods of RNA extraction from the cladocerans are still lacking. This study evaluates the extraction of RNA from tropical freshwater cladocerans Moina micrura using two methods: the phenol-chloroform extraction method (QIAzol) and a column-based kit (Qiagen Micro Kit). Glycogen was introduced in both approaches to enhance the recovery of extracted RNA and the extracted RNA was characterised using spectrophotometric analysis (NanoDrop), capillary electrophoresis (Bioanalyzer). Then, the extracted RNA was analysed with reverse transcription polymerase chain reaction (RT-PCR) to validate the RNA extraction method towards downstream gene expression analysis. The results indicate that the column-based kit is most suitable for the extraction of RNA from M. micrura, with the quantity (RNA concentration = 26.90 ± 6.89 ng/μl), quality (A260:230 = 1.95 ± 0.15, A280:230 = 1.85 ± 0.09) and integrity (RNA integrity number, RIN = 7.20 ± 0.16). The RT-PCR analysis shows that the method successfully amplified both alpha tubulin and actin gene at 33-35 cycles (i.e. Ct = 32.64 to 33.48). The results demonstrate that the addition of glycogen is only suitable for the phenol-chloroform extraction method. RNA extraction with high and comprehensive quality control assessment will increase the accuracy and reliability of downstream gene expression, thus providing more ecotoxicological data at the molecular biological level on other freshwater zooplankton species.