Vinayak Mohandas creator Swapna Alex (Guide) text Vellayani Department of Plant Biotechnology, College of Agriculture 2022 monographic 78p. The study entitled “Silver, magnesium and zinc nanoparticles for improving the efficiency of RT-qPCR” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2019-2021. The objective of the study was to evaluate the efficacy of silver, magnesium and zinc nanoparticles in improving the efficiency of RT-qPCR. Spike samples of black pepper var. Panniyur-1 and leaf samples of rice var. Uma for nucleic acid isolation were collected from Instructional Farm, College of Agriculture, Vellayani. Efficacy of nanoparticles for improving efficiency of PCR/RT-qPCR were analyzed using housekeeping gene Actin in rice and black pepper and low copy genes OsYUCCA1 (flavin monooxygenase) in rice and TAA1 (tryptophan amino transferase) in black pepper. Primers were designed using Primer Express software for Actin1 and OsYUCCA1 in rice and reported primers were used for Actin and TAA1 in black pepper. For PCR, DNA was isolated from rice using modified Cetyltrimethylammonium bromide (CTAB) method and the good quality was confirmed using nanodrop spectrophotometer. Different concentrations (1 mgL-1 to 250 mgL-1 ) of silver (Ag), zinc oxide (ZnO) or magnesium oxide (MgO) nanoparticles (NPs) (Sigma Aldrich, USA) or their combinations were included in PCR reaction mix and PCR was performed at 95oC for 2 min followed by 35 cycles of 95oC for 15s, 48-55oC for 1 min, 72oC for 45s and final extension at 72oC for 5 min. A control was kept without any nanoparticles. Three replications were done. Efficiency and specificity of PCR were checked by comparing the intensity of the expected amplicon (213 bp) in agarose gel electrophoresis using Image lab software. Inclusion of Ag NPs and MgO NPs in PCR reaction mix at concentrations of 4 mgL-1 and 175 mgL-1 respectively, exhibited 2.3-fold and 5.7-fold increase in intensity of band. ZnO NPs at a concentration of 175 mgL-1 showed an amplification comparable to that of control (1.08-fold). Simultaneous inclusion of both Ag NPs and MgO NPs at 76 concentrations of 2 mgL-1 and 175 mgL-1 respectively in conventional PCR exhibited 6.3- fold increase in intensity of band. RNA was isolated from rice and black pepper using Trizol method and converted to cDNA. The quality of synthesized cDNA and the specificity of the primers were checked by PCR using cDNA followed by agarose gel electrophoresis. Specific amplicons of size 213 bp, 190 bp and 266 bp were obtained for Actin1 (rice), Actin (pepper) and TAA1 respectively. RT-qPCR using SYBR Green dye-based assay was carried out by including Ag (4 mgL-1 ), MgO (175 mgL-1 ) or ZnO (175 mgL-1 ) nanoparticles or their combinations in different concentrations based on the results of PCR. Technical replicates were maintained and 40 cycles of RT-qPCR was carried out at 95oC for 15s, 48-60oC for 15s and 60oC for 45s. PCR efficiency was analyzed using LinReg software. Cq values of RTqPCR were reduced in housekeeping genes and low copy number genes with inclusion of MgO nanoparticles. Among the treatments tried, MgO NPs showed maximum fold increase in amplification in all the genes analyzed (60.84, 13.70, 6.64 and 1.36-fold in Actin1, OsYUCCA1, Actin and TAA1 respectively). Combination of Ag NPs and MgO NPs exhibited 0.39-to-17.70-fold increase in amplification in RT-qPCR. Zinc oxide nanoparticles showed inhibition of amplification. To conclude, combination of Ag NPs and MgO NPs at concentrations of 2 mgL-1 and 175 mgL-1 respectively exhibited maximum improvement (6.3-fold increase) of conventional PCR efficiency. Addition of MgO NPs at a concentration of 175 mgL-1 exhibited maximum fold increase in RT-qPCR amplification (1.36 to 60.84-fold) in housekeeping and low copy number genes. Concentrations of MgO NPs and their combination with Ag NPs can be further optimized for improving PCR and RT-qPCR efficiency, especially for diagnostic detection purposes. MSc Plant Biotechnology Nanoparticles Silver, magnesium and zinc 660.6 VIN/SI PG https://krishikosh.egranth.ac.in/handle/1/5810221539 https://krishikosh.egranth.ac.in/handle/1/5810221539