METHOD OPTIMIZATION OF DIATOM NAVICULA SALINICOLA NLA TOTAL RNA ISOLATION FOR TRANSCRIPTOMIC APPLICATION

• Main Author: Fitria, Murni
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/49589
• Institution: Institut Teknologi Bandung

The main problem on RNA extraction and analysis are the instability of the RNA due to its single strand structure, the presence of reactive hydroxyl group at the 2’ carbon of the ribose, the exposure of RNase during extraction or analysis process, the contamination of carbohydrates, protein, and lipids cell that could be potentially extracted with the RNA. Whereas, the genetic information stored in RNA or transcriptome can be the basis for studying metabolism, as the metabolism of photosynthetic pigments of diatom which are grown in a low or high light intensity. The aim of this study was to obtain the optimum method of isolating total RNA from local diatom which is able to provide RNA isolate that is appropriate for transcriptomic analysis. The research stages included screening diatom single cell and culturing it until a pure cell culture was obtained, developing cell growth curve, isolating of genomic DNA, identifying the diatom sample based on morphology of cells and homology 18S rRNA-V4, isolating total RNA, and optimizing RNA isolation method. Results showed that the diatom sample having cell shape like a surfboard with the length between 10–20 µm have been succeed to be screened and cultivated. The cell biomass of diatom Navicula sp. NLA harvested at the exponential phase had an increase of average cells density about 39 times from the start, i.e from 100 cell/mL to 3.94 million cells/mL. The electrophoresis analysis showed, a genomic DNA ribbon longer than 10.000-bp. Analysis of 476-bp 18S rRNA-V4 nucleotide sequence confirmed that the diatomic sampel is identic to Navicula salinicola, therefore it wasnamed as Navicula salinicola NLA. The total RNA isolation method that gave the most optimal result from Navicula salinicola NLA was the modified method of TrizolTM (ThermoFisher Scientific). The total RNA yield was high (64,805 µg) with the value of A260/230 and A260/280 each higher than 2,0. These results meet the criteria for good quality RNA suitable for transcriptomic analysis, namely a high level of purity and the complete RNA distinctive bands observed electrophoretically.