xxpress RT-qPCR to Analyse the Quality and Quantity of cDNA

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xxpress RT-qPCR to Analyse the Quality and Quantity of cDNA

A recent article by the Queensland University of Technology investigated candidate genes for facilitating freshwater adaptation in palaemonid prawns. These genes were identified and their expression validated in the model species M. koombooloomba.


Background: The endemic Australian freshwater prawn, Macrobrachium koombooloomba, provides a model for exploring genes involved with freshwater adaptation because it is one of the relatively few Macrobrachium species that can complete its entire life cycle in freshwater.

Methods: The present study was conducted to identify potential candidate genes that are likely to contribute to effective freshwater adaptation by M. koombooloomba using a transcriptomics approach. De novo assembly of 75 bp paired end 227,564,643 high quality Illumina raw reads from 6 different cDNA libraries revealed 125,917 contigs of variable lengths (200–18,050 bp) with an N50 value of 1597.


The quality and concentrations of each cDNA library was assessed using Bioanalyzer (Agilent 2100, version 6), Qubit R 2.0 Fluorometer (Invitrogen, Life Technologies) and RT-qPCR (XXPress Thermal Cycler, BJS Biotechnologies, UK).

Results: In total, 31,272 (24.83%) of the assembled contigs received significant blast hits, of which 27,686 and 22,560 contigs were mapped and functionally annotated, respectively. CEGMA (Core Eukaryotic Genes Mapping Approach) based transcriptome quality assessment revealed 96.37% completeness. We identified 43 different potential genes that are likely to be involved with freshwater adaptation in M. koombooloomba. Identified candidate genes included: 25 genes for osmoregulation, five for cell volume regulation, seven for stress tolerance, three for body fluid (haemolymph) maintenance, eight for epithelial permeability and water channel regulation, nine for egg size control and three for larval development. RSEM (RNA-Seq Expectation Maximization) based abundance estimation revealed that 6,253, 5,753 and 3,795 transcripts were expressed (at TPM value ?10) in post larvae, juveniles and adults, respectively. Differential gene expression (DGE) analysis showed that 15 genes were expressed differentially in different individuals but these genes apparently were not involved with freshwater adaptation but rather were involved in growth, development and reproductive maturation.

Discussion: The genomic resources developed here will be useful for better understanding the molecular basis of freshwater adaptation in Macrobrachium prawns and other crustaceans more broadly.


Read the full article here: Candidate genes that have facilitated freshwater adaptation by palaemonid prawns in the genus Macrobrachium

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