A Combined RNA Preservation and Extraction Protocol for Gene Expression Studies in Cacao Beans

Abstract

Despite the high economic importance of cacao beans, few RNA-based studies have been conducted on this plant material and hence no optimal RNA-extraction has been reported. Moreover, extraction of high-quality RNA from recalcitrant cacao bean tissue has shown many difficulties and requires optimization. Furthermore, cacao beans are mostly found at remote and under-resourced locations, which pressures the outsourcing of such analysis and thereby demands RNA-stable preservation and transportation of cacao beans. This study aims to select an appropriate RNA extraction and preservation/ transportation method for cacao beans. For this purpose, three sample homogenization and five extraction protocols on cacao beans were compared. In addition, 13 preservation conditions-differing in tissue crushing degree, preservation method, duration, and temperature-were compared and evaluated. A comparative analysis revealed that CTAB-based homogenization and extraction outcompeted all tested commercial protocols in RNA yield and integrity, respectively. Preservation at −80°C affected RNA quality the least, whereas freeze-drying was most suitable for transportation at room temperature for maximum 1 week. The cacao bean RNA obtained from the selected methods were compatible for downstream applications. The results of this study will facilitate on-field sampling and transportation of genetically sensitive cacao material prior to cacao bean transcriptomic studies. In addition, valuable insights on sample homogenization, extraction, preservation, and transportation have been provided, which is of interest to every plant geneticist.

Selection and validation of reference genes for accurate RT-qPCR gene expression normalization in cacao beans during fermentation

“…The pH of the fermenting heap was measured daily using a Hanna Edge Multi-parameter pH meter (HI2020-01). All samples were immediately retained on dry ice after collection, then freeze dried (− 40°C, 0.25 bar for 24 h, Heto Powerdry pl3000, Thermo Fisher Scientific), shipped to Belgium and stored at − 80°C until needed (conform with the methods described by De Wever et al (2020)).…”

“…Extraction of RNA was conducted according to a protocol initially described by Luypaert et al (2017) and slightly adapted for the extraction of RNA specifically from cacao beans by De Wever et al (2020). Briefly, 60 mg of cryogenically crushed cacao bean material was homogenized with 700 μl at 65°C preheated extraction buffer [3% (w/v) CTAB; 100 mM Tris-HCl (pH 8.0); 1.4 M NaCl; 20 mM Na2 EDTA; 5% (w/v) PVP and 1% (v/v) β-mercaptoethanol (added just before use)].…”

“…Before use, the obtained RNA was DNase treated using the RapidOut DNA Removal kit (ThermoFisher Scientific, Waltham, MA, USA) according to manufacturer's protocol. Next, several RNA extraction parameters, i.e., RNA quantity, purity, and integrity, were determined as described by De Wever et al (2020). The RNA quantity was measured by the Qubit RNA HS assay kit and the Qubit® 2.0 Fluorometer (Invitrogen, Carlsbad, CA); RNA purity was measured by A260/280 (protein contamination) and A260/230 (polyphenol and polysaccharide contamination) using the NanoDrop (ND) 1000 UV-Vis spectrophotometer (NanoDrop Technologies, USA).…”

“…The generated data was analyzed using PROsize 2.0® (Agilent), which resulted in RNA quality numbers (RQN). The retrieved chromatograms were in addition scored visually from A to F, representing high and low integrity, respectively according to De Wever et al (2020).…”

“…cDNA synthesis and RT qPCR cDNA synthesis and qPCR analysis were conducted as described by De Wever et al (2020). cDNA was synthesized using the iScript™ Advanced cDNA Synthesis kit (BioRad, Richmond, CA, USA) according to manufacturers protocol.…”