AN OPTIMISED METHOD FOR EXTRACTION OF HIGH-QUALITY RNA FOR RNA-SEQ FROM AMAZONIAN TREES

Abstract

The study aimed to develop a robust and reliable method to extract ribonucleic acid (RNA) from Guazuma crinita and Calycophyllum spruceanum woody tissues for RNA sequencing. Thus, 60 plants of each species were obtained, and four RNA extraction methods were evaluated (Direct-zol^TM kit, TRIzol, cetyltrimethylammonium bromide (CTAB) and CTAB with Direct-zol^TM kit). Subsequently, six samples for each species were selected for sequencing. As a result, the CTAB method combined with TRI-reagent and Direct-zol^TM RNA kit obtained RNA of good quality in both species, as the RNA integrity number (RIN) values were higher than 7.9 in all samples. Inhibitors were removed in G. crinita with polymerase chain reaction (PCR) inhibitor removal kit. The sequencing process, on average per sample, were 4,559,130 kbp in 45,139,903 reads with a Q20 of 98.49% and Q30 of 95.59% obtained for G. crinita and 5,510,902 kbp in 54,563,395 reads with a Q20 of 98.23% and Q30 of 94.94% obtained for C. spruceanum. Thus, a robust method was developed, enabling the extraction of RNA in both quality and quantity for RNA-seq experiments, successfully overcoming the challenges posed by inhibitors and high concentrations of exudates, alkaloids, polysaccharides and phenols.