cDNA from Total RNA of Navicula salinicola NLA Diatom Grown Indoor and Outdoor for sqRT-PCR Applications Trisha Audria, Sari Dewi Kurniasih Indrawan, Alfredo Kono, Yanti Rachmayanti, Zeily Nurachman*
Biochemistry Division, Faculty of Mathematics and Natural Science, Institut Teknologi Bandung, Jalan Ganesa 10, Bandung 40132, Indonesia
*zeily[at]itb.ac.id
Abstract
Navicula salinicola NLA, a potential biodiesel feedstock, shows light intensity-dependent lipid metabolism. However, knowledge about lipid metabolism pathways in N. salinicola NLA under different light conditions is lacking. This study aimed to obtain cDNA from N. salinicola NLA RNA for sqRT-PCR analysis. Methods included indoor/outdoor cultivation, total lipid isolation, primer design for internal gene control (rbcL and ACTB), genomic DNA isolation, primer compatibility testing, total RNA isolation, cDNA synthesis, and evaluation of control internal genes on N. salinicola NLA cDNA. The results shows that N. salinicola NLA cells had boat-like shapes, with dark brown (indoor) and light yellowish brown (outdoor) cultures. Initial cell density was 0.75 x 10^6 cells mL^-1, increasing to 4.1 x 10^6 cells mL^-1 (indoors) and 2.2 x 10^6 cells mL^-1 (outdoors) after 6 days. This resulted in specific growth rates of 0.33 +- 0.02 and 0.24 +- 0.03 x 10^4 cells mL^-1 day^-1, doubling times of 2.11 +- 0.12 and 2.94 +- 0.47 days, biomass productivity of 0.53 +- 0.01 and 0.23 +- 0.01 mg mL^-1 day^-1, and yields of 0.56 +- 0.08 and 0.41 +- 0.04 g L^-1 on the sixth day. Indoor cultures yielded lipids at 41.12%, while outdoor cultures yielded 46.1%. Internal control genes (NAVsa-RbcL-1 and NAVsa-ACTB-1) were identified from a 500 bp segment, demonstrating primer suitability. Total RNA was recovered during exponential and stationary phases, showing bands at 1400, 900, and 250 bp (indicating 28S, 18S, and 5S RNA) with a concentration range of 800-100 ng L^-1. ACTB was consistently expressed in both indoor and outdoor cultures.
