Effect of DNase treatment on RNA extraction from preimplantation murine embryos.

The quality of RNA is crucial when performing microarray experiments. This is particularly important when dealing with preimplantation embryos, from which a minimum yield of RNA of good quality can be produced. We report the optimization of several RNA extraction methods applied to preimplantation embryos at different stages of development. The quality of the samples was confirmed using a microarray and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. A total of 30 cultured two-cell stage embryos of ICR mice were pooled at the 8-cell, morula, and blastocyst stages. The embryos were divided into two groups comprising DNase-treated and non-DNase-treated RNA samples. Total RNA was extracted using a Pico Pure RNA Isolation Kit following the manufacturer protocol, with some modifications. Lysed samples were bound to a silica-based filter, treated with deoxyribonuclease I (DNase I), and washed several times before elution. RNA concentration and integrity were evaluated using an Agilent 2100 Bioanalyzer and an RNA 6000 Pico Assay kit. Although concentrations of non-DNase-treated RNAs were higher than DNase-treated RNA, DNase-treated RNA gave a higher RNA integrity number compared with non-DNase-treated RNA. Inclusion of DNase treatment in the RNA extraction procedure gave the best quality RNA samples from preimplantation embryos, as validated by microarray and RT-qPCR quality control.

Nutritional content and in vitro antioxidant potential of Garcinia atroviridis (Asam gelugor) leaves and fruits.

INTRODUCTION: The objective of this study was to determine antioxidant potential of Garcinia atroviridis leaves and fruits extracts in vitro. METHODS: Antioxidant activity was assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. Total phenolic content (TPC) of the extracts was estimated as gallic acid equivalent by Folin-Ciocalteau method. Proximate analysis was determined based on the Association of Official Analytical Chemists (AOAC) procedures. RESULTS: Garcinia atroviridis leaves extracted at 100 degrees C/15 min demonstrated the highest TPC value (21.21 +/- 0.28 mg GAE/mg) and was significantly different (p < 0.05) from that of leaves extracted at 60 degrees C/6 h and 40 degrees C/12 h. On the other hand, the fruit extracted at 60 degrees C/6 h showed the highest TPC value (16.23 +/- 0.18 mg GAE/mg) (p < 0.05) compared to the fruit extracted at 40 degrees C/12 h and 100 degrees C/15 h respectively. The antioxidant activities of both samples were positively correlated with the TPC values based on DPPH-radical-scavenging activity and ferric reducing power estimation. Garcinia atroviridis leaf extract contained significantly higher proteins, carbohydrate and ash contents (2.16% +/- 0.08; 15.98% +/- 0.12 and 0.72% +/- 0.07 respectively) than its fruit extract (0.46% +/- 0.08, 8.64% +/- 0.06 and 0.15% +/- 0.06) respectively). The energy content was also found to be higher in the leaf (73.64% +/- 2.15) compared to the fruit (38.38% +/- 1.72) (p < 0.05). CONCLUSION: The findings indicate that G. atroviridis leaves and fruits have potential for use as a source of natural antioxidants and nutrients for therapeutic purposes against free radical mediated health conditions.