Lipase-I gene; cDNA; Kasalath rice; Indonesian Black rice; RNA extraction; reverse transcription; PCR amplification; lipase inactivation
Lipase is responsible for hydrolytic rancidity of processed rice. It hydrolyzes lipids by breaking intra-chain ester bonds into free fatty acids. This experiment aimed to extract RNA from Kasalath and Indonesian Black (IDB) rice lines and determine genes responsible for lipase activity. The practical started by first extracting all RNAs from leaves and roots of Kasalath and IDB rice lines. The RNAs were then converted to cDNA by reverse transcription. cDNA produced from the reaction was then amplified using forward and reverse primer targeting lipase gene. Amplified products were then verified using agarose gel electrophoresis. The total RNA concentrations were found to be 197.28ng/µL and 25.84ng/µL respectively for leaf and roots tissues of Kasalath rice, while 197.44ng/µL and 29.28ng/µL respectively for leaf and roots tissues of IDB rice. Three bands were observed for each Kasalath leaf (516bp, 422bp and 322bp) and roots (565bp, 422bp and 322bp). Three bands (598bp, 485bp and 347bp) were found for both IDB leaf and roots.
In conclusion, leaf had higher concentrations of total RNA than roots in both rice lines, indicating that lipase expression was higher in leaf. Three bands (of which two were contaminants) found in both leave and roots tissues of Kasalath and IDB rice lines. The bands 516bp and 565bp corresponded to lipase-I cDNA in Kasalath leave and roots respectively while IDB's leave and roots were both 598bp. The lipase-I cDNA was generally longer for IDB than Kasalath. Contamination of samples by DNA was suspected so further analysis had to be conducted to validate findings as experiment was compounded with errors.
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[...] Three bands were observed for both leaf and roots samples of Kasalath and IDB rice lines. This was not consistent with expected result. Theoretically, while total RNA was reverse transcribed to cDNA, only specific cDNA corresponded to lipase enzyme would be selectively amplified by PCR. Therefore, only one band should be expected in gel image. Presence of additional bands suggested genomic DNA (gDNA) was not completely removed from test sample. As lipase gene locus in gDNA had high homology to synthesized cDNA, PCR primers could also amplify the genomic sequences leading to additional products. [...]
[...] Results Quantification of RNA using Nanodrop Table Absorbance at 260nm and 280nm, RNA concentration and A260:A280 ratio for leaves and roots samples of Kasalath rice. Note: Data was obtained from G7. Table Absorbance at 260nm and 280nm, RNA concentration and A260:A280 ratio for leaves and roots samples of Indonesian Black rice. Calculations to prepare 1µg total RNA for reverse transcription For Kasalath leaves: Volume of 1µg total RNA = (1µg x 1µL) 197.28 ng/µL = 5.069 µL Similar calculations were applied for Kasalath roots as well IDB leaves and roots samples. [...]
[...] In conclusion, leaf had higher concentrations of total RNA than roots in both rice lines, indicating that lipase expression was higher in leaf. Three bands (of which two were contaminants) found in both leave and roots tissues of Kasalath and IDB rice lines. The bands 516bp and 565bp corresponded to lipase-I cDNA in Kasalath leave and roots respectively while IDB's leave and roots were both 598bp. The lipase-I cDNA was generally longer for IDB than Kasalath. Contamination of samples by DNA was suspected so further analysis had to be conducted to validate findings as experiment was compounded with errors. [...]
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