3.5.1 DEPC Treatment
All equipment and consumables were treated with Diethylpyrocarbonate (DEPC) water to inhibit RNase enzyme activity prior to total RNA extraction.
Equipment and consumables such as microcentrifuge tubes, micropipette tips, and tip boxes were immersed in 0.1 % of DEPC water for overnight. It was then dried in the oven and sent for autoclave.
Liver
27 3.5.2 RNA Extraction from Liver Samples
GENEzolTM TriRNA Pure Kit was used to extract total RNA from mice liver of all treatment groups. The extraction procedures were carried out according to the manufacturer protocol. First of all, approximately 30 mg of liver tissue was placed into a 1.5 mL microcentrifuge tube added with 350 µL of GENEzolTM Reagent and the tissue was grinded completely using a micropestle. It was then incubated at room temperature for 5 minutes Next, the sample was centrifuged at 16,000 x g for 1 minute and the supernatant was transferred to a new 1.5 mL RNase-free microcentrifuge tube. Equivalent amount (1:1) of absolute ethanol was directly added to the sample mixture in GENEzolTM Reagent. The mixture was then vortexed to mix well and a RB column was placed in a 2 mL Collection Tube. Approximately 700 µL of the sample mixture was transferred to the RB column and centrifuged at 16,000 x g for 1 minute, and the flow-through was discarded.
Approximately 400 µL of Wash Buffer was pipetted to the RB Column and centrifugation was run at 16,000 x g for 30 seconds. Next, the flow-through was discarded and the RB Column was then put back in the Collection Tube.
Preparation of DNase I solution was done in a 1.5 mL RNase-free microcentrifuge tube by adding 5 µL of DNase I and 45 µL of DNase I Reaction Buffer and mixed well by gentle pipetting. DNase I solution was then added into the centre of the RB column matrix and incubated for 15 minutes at room temperature. Approximately 400 µL of Pre-Wash Buffer was added to the RB Column and centrifuged at 16,000 x g for 30 seconds. Next, the flow-through
28 was poured out and the RB Column was put back in the Collection Tube and pipetted 600 µL of Wash Buffer into the RB Column followed by centrifugation at 16,000 x g for 30 seconds. The flow-through was then poured out and the RB Column was placed back in the Collection tube. The tube was then centrifuged at 16,000 x g for 3 minutes to dry the column matrix.
The dried RB Column was then placed in a new and clean 1.5 mL RNase-free microcentrifuge tube and pipetted carefully 50 µL of RNase-free Water to the centre of the column matrix and let stand for 3 minutes to make sure the RNase- free Water was absorbed fully by the matrix. Lastly, the tube was centrifuged at 16,000 x g for 1 minute to elute the purified RNA. The RB Column was discarded and the microcentrifuge tube containing the purified RNA was labelled properly and stored in -20 °C for further use.
3.5.3 Quantification of RNA Samples
The extracted RNA samples were quantified by measuring the concentration and purity of RNA at wavelength of 260 nm and 280 nm using the NanoDrop 2000 Spectrophotometer available in the lab of Biotechnology unit, UTAR.
Prior to loading the RNA sample onto the optical measurement surface, the measurement surface and lid were cleaned with ethanol sprayed lint-free Kimwipe paper. Next, a volume of 1 µL nuclease-free water was used as blank, and 1 µL of RNA sample was carefully pipetted onto the centre of optical measurement surface and the lid was closed. The sample was analysed, and the nucleic acid concentration and purity readings were recorded. Both the optical
29 measurement surface and lid were wiped before it was used for the subsequent RNA sample.
The purity of the RNA sample was assessed by measure the absorbance ratio at 260 nm and 280 nm (A260/A280). The RNA sample with ratio values of 2.0-2.2 was generally considered as high RNA purity (Carpinetti et al., 2021). Ratio value higher than 2.2 might be due to changes in sample acidity by basic solution whereas ratio value that was lower than 1.9 might indicate changes in sample acidity by acidic solution or contamination by residual phenol or protein (DeNovix, 2019).
3.5.4 Qualification of RNA Samples
The quality and integrity of the extracted total RNA samples was determined by running gel electrophoresis. Agarose powder and 1X Tris-Borate-EDTA (TBE) buffer were used to prepare 1 % agarose gel. Approximately 0.2 g of agarose powder was poured into 20 mL of 1X TBE buffer in a conical flask. The mixture was mixed well by gentle swirling and placed in a microwave to heat the mixture and dissolve the agarose powder completely in TBE buffer. A clean 0.75 mm comb was placed in a gel casting tray and the cooled mixture was poured into the gel casting tray fixed with the comb. The bubbles formed was removed by using pipette tip. The gel was left at room temperature for around 30 minutes to solidify. Next, the comb was gently removed, and the gel was carefully put in the gel electrophoresis tank. After that, 1X TBE buffer was
30 added into the tank until it covered the whole gel to a depth of approximately 1 mm.
Before loading the RNA samples into the wells, 1 µL of Novel Juice was mixed with 5 µL of RNA samples on a clean parafilm. A volume of 2 µL 1 kb DNA ladder was also mixed with 1 µL of Novel Juice and loaded into the first and last well of gel. The gel was then run at 90 V for 35 minutes and subsequently viewed using Bio-rad Gel Documentation System under UV transilluminator.
Distinguishable bands of 28S and 18S rRNA were visible in the RNA gel image.
RNA gel image with no obvious smearing and presence of high brightness 28S and 18S rRNA indicate good RNA integrity (Carpinetti et al., 2021).