OPTIMIZATION OF RNA EXTRACTION METHOD FROM TROPICAL MARINE NANNOCHLOROPSIS OCEANICA GSI TO OBTAIN GOOD QUALITY EXTRACT FOR TRANSCRIPTOMICS ANALYSIS

• Main Author: Nayassa, Andita
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/45531
• Institution: Institut Teknologi Bandung

Nannochloropsis is a genus of unicellular microalgae which has 7 species group, one of which is the marine microalgae Nannochloropsis oceanica. Many studies have been done on marine microalgae Nannochloropsis, among which reported Nannochloropsis oceanica as a potential microalgae due to high triacillgliserol (TAG) content, rapid growth, and easy to grow in laboratory. Morphologically Nannochloropsis oceanica cells are observed to be round in shape, green in colour,2-5p.m in diameter, and throbbing. Transcriptomics is a study of transcriptomes that requires good quality RNA extract as an analytical material, namely RNA extract with minimum concentration of 133.33 ng/ p.L in total volume of 30 p.L or equivalent to the RNA amount of at least 4,000 ng, the presence of large RNA in an intact, and high purity of extract is indicated by A260I2BO and A2601230 ratio of at least I.8 respectively. A standard or specific method for isolation of RNA from Indonesian strains of Nannochloropsis oceanica are not available yet. This study is aimed to optimize the RNA isolation method from microalgae especially from Indonesian Nannochloropsis oceanica GSI strain is harvested in two different growth phases, namely the middle exponential phase and the stationary final phase. This optimization is targeted to obtain total RNA extract from Indonesian marine Nannochloropsis oceanica GSI with good quality making it feasible for transcriptomic applications. The optimized method was TR!sure TM protocol from Bioline, which was modified in several ways, including: (i) the use of microalgae cell pellets up to a maximum of 0. 7 grams in order to increase the concentration of final RNA extract, (ii) the removal of a brownish-white layer covering the microalgae cell pellets (after centrifugation) in order to raise the ratio value A260'2BO , (iii) the addition of beta-mercaptoethanol together with TR!sure TM or GENEzol™ to isolate large RNA fragments of 1000 bp and 1500 bp, (iv) the four times washing by ethanol in RNA pellets in order to increase the A26omo ratio. The results obtained indicated that the RNA extract could be suitable for transcriptomics due to its high concentration (24I.4- 807. I ng/ p.L or total RNA extract of 7,242 ng-24,213 ng), A260!2so ratio value of I.8I- 1.98 and A260!230 ratio value of 1.84-2.24, and the presence of large RNA .fragments of 1500 bp and 1000 bp in intact condition (not degraded).