Optimization of the CTAB3-LICL and commercial kit methods in the process of RNA isolation and amplification in strawberries fruit (Fragaria spp.)

Abstract

RNA (Ribonucleic Acid) isolation is a basic technique in the field of molecular biology. The purpose of RNA isolation is to acquire pure mRNA that can be used to see the expression of certain genes in an individual. Many methods can be used to perform RNA isolation, both conventional and modern methods. The purpose of this study were to compare the results of total RNA isolated using the CTAB3-LiCl (conventional) method and the Commercial Kit (modern) method and then amlification of FaPYR1 and FaCHS genes from mRNA templates in the strawberry fruits. The RNA isolation done by using the Kit method referring to the Geneaid "RNA Total Mini Kit (Plant)" kit protocol. In general, the RNA isolation may consist of three stages: (1) Lysing the membrane or cell wall by physical and chemical destruction, (2) Removing unwanted components such as proteins, polysaccharides, and cell debris, (3) Searching for pure RNA by precipitation. The total RNA isolates were analyzed quantitatively by TECAN nanodrop spectrophotometer to determine the purity and concentration of RNA. Biorad thermal cylers used for amplification for the target genes. The results showed that the RNA isolated using the CTAB3-LiCl method had a higher RNA concentration than the Kit method, and the purity produced from the two methods did not differ significantly. Thus, in this research, the total RNA isolated using the CTAB3-LiCl method is more optimal than the Kit method. The FaPYR1 and FaCHS genes amplificated in 627 bp, Actin gene in 262 bp, and 26S-18S Housekeeping gene in 146 bp to all samples selected.