Evaluation of Passiflora edulis Leaf Sample Storage Methods on RNA Quality and Suitability for Use in RT-PCR Assays

Abstract

It is prerequisite to extract Ribonucleic Acid (RNA) with high quality and integrity in order to carry out molecular biology studies in any plant species. Samples collected from remote fields require preservation before being processed for RNA extraction and downstream process like RT-PCR, real time PCR and genome-wide expression studies. It is therefore important to identify efficient and reliable sample storage methods that stabilize RNA, protecting it from the activities of RNAse in intact samples before analysis. This study was designed to evaluate the effect of different storage conditions for passion fruit leaves on RNA quality and suitability for RT-PCR for two time-points; one week and two weeks post-harvest. Passion fruit leaf samples with suspected viral symptoms were collected from the field and stored using FTA® cards, RNAlater solution, cold ice followed by transfer to -80°C freezer, drying on silica gel and drying in between the sheets of newsprints (as herbarium). The samples were kept for 1 and 2 weeks before RNA extraction and subsequent semi-quantitative RT-PCR to amplify the housekeeping genes AtropaNad and Cowpea Aphid Borne Mosaic Virus (CABMV); one of the major viruses causing  passion fruit woodiness  disease in Kenya. Good RNA yield and quality were obtained from samples stored in silica gel for 1 and 2 weeks after collection similar to -80°C frozen samples a choice preservation method by many laboratories all over the world. Further results confirmed that RNA extracted from samples stored in silica gel was fit for RT-PCR amplification. This study shows that RNA of good yield and quality that is useful for downstream applications can be obtained from passion fruit leaf samples stored in silica gel.