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RNA extraction and amplification of putative lipase gene from Kasalath and Indonesian Black rice

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  1. Introduction
  2. Extraction and quantification of total RNA
  3. Quantification of RNA using Nanodrop
  4. Discussions
  5. Conclusion

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.

[...] Xing Zhao Yu Zhang Guo L & Miao C 2005, ?Study on gene differential expression in roots and leaves between hybrid CR147 and its parents at seedling stage', Scientia Agricultura Sinica, vol no pp. 1275-1281. Vijayakumar KR & Gowda LR 2012, ?Temporal expression profiling of lipase during germination and rice caryopsis development', Plant Physiology and Biochemistry, vol pp. 245-253. Wijer DBMAW, Zee AHM, Boer Belitse SV, Kroon AA, Leeuw PW, Schiffers Janssen RGJH, Duijn CM, Stricker BHCH & Klungel OH 2009, ?Determinants of DNA yields and purity collected with buccal cell samples', European Journal of Epidemiology, vol pp. 677-682. [...]


[...] 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. [...]


[...] Agarose gel concentration should be increased to as recommended by DNA ladder company provider (Thermo Scientific 2013). Lower volume of DNA ladder should be used to prevent overloading. 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. [...]


[...] Conventional study of lipase focused on isolation and characterization of this undesirable enzyme, followed by invention of chemical and heat treatment workflow to inactivate its activity. Despite commonly practiced in rice processing industry, these treatments lack capability to completely rid the rice grains of lipase presence (Silva et al. 2006). Through massive advances in biotechnology, expanding knowledge on rice plant genome paves way to more promising approaches to inhibit lipase on its molecular level. Reverse transcription is a process that converts RNA to cDNA. [...]


[...] The observations were consistent with expected results where concentration of RNA was higher in leaf than in root. Xing et al. (2005) reported that the differential expression of genes were richer in lead than in roots. The total RNA concentration initially served as an indicator of expression level of lipase. The higher the expression, the more lipase mRNA was produced. Following seeds germination, the lipase I expression transcript in root increased linearly but declined after a few days. In contrast, due to the physiological implications of lipase I in driving shoot elongation through mobilization of fats to sustain normal cellular metabolism, its transcripts' expression increased linearly in shoot and did not show signs of reduction (Vijayakumar & Gowda 2012). [...]

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