Quoted from WHO, Indonesia is one of the tropical countries affected by the Dengue Virus (DENV). The emerging dengue vectors have four antigenically distinct serotypes. The four dengue virus serotypes were DENV-1, DENV-2, DENV-3 and DENV-4. Dengue virus is transmitted by Aedes aegypti and Aedes albopictus mosquitoes. Dengue virus is transmitted through the bite of a female Aedes mosquito that occurs when a mosquito sucks the blood of a person infected with the dengue virus and the mosquito bites another person. Clinical symptoms include Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS). There are approximately 100 million cases of Dengue Fever (DF), 500,000 cases of DHF and 25,000 deaths reported annually worldwide. Therefore, further research is needed on this dengue virus.
Dengue virus is an RNA virus that can be identified using the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and Polymerase Chain Reaction (PCR) methods. The success of RT-PCR and PCR is determined by the RNA extraction process; with the right precipitation solvent to obtain high purity RNA. The solvents used were Isopropanol, DMSO, 96% Ethanol, Methanol, Acetone-Methanol (1:1), DMF, and distilled water. In this study, TRIzol was used as a method of RNA extraction in dengue virus DENV 1 Surabaya Genbank: AB915377.
RNA extraction using the TRIzol method used the TRIZol reagent consisting of phenol monophase solution and guanidium isocyanate (Rio et al., 2010). DENV-1 sample was dissolved with TRIZol reagent then chloroform was added to cause phase separation. The organic phase contains protein; DNA is at the interface and the clear phase contains RNA. The clear phase was transferred to a new tube and then added the precipitation solution, namely: isopropanol, DMSO, 96% ethanol, methanol, methanol acetone (1: 1), DMF, and distilled water. In solution, guanidium (NH2) 2C = NH2 + from the TRIzol reagent neutralizes the negative charge on the PO43- group of nucleic acids and makes the nucleic acids hydrophobic and less soluble in water so that they experience precipitation.
Precipitation solvents which have a low dielectric constant have the ability to support the interaction between guanidium and phosphate ions so that the nucleic acids become less hydrophilic and precipitate. RNA purity was determined by measuring the absorbance at a ratio of 260/280 nm with a nano drop spectrophotometer. In this study, the absorbance of RNA with isopropanol precipitation solvent was 1.78, DMSO was 1.36, 96% ethanol was 1.54, methanol was 1.01, acetone-methanol (1: 1) was 1.81, DMF was 1.70, and distilled water is 1.40.
Pure RNA has an absorbance value of 1.8 – 2.1 (Farrel., 2005). Pure RNA was obtained in the isolation process with acetone-methanol (1: 1) precipitation solvent with an absorbance value of 1.81. The RNA isolate that has been obtained is then reversed transcription with RT-PCR to obtain c-DNA (complementary DNA) and then amplified. The amplified c-DNA was then electrophoresed with agarose gel and visualized using a UV Trans-illuminator.
The dengue virus is carried by female mosquitoes of the Aedes aegypti and Aedes albopictus species. This viral RNA can be identified by using the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and Polymerase Chain Reaction (PCR) methods. The modification of the RNA isolation method from DENV-1 with the TRIzol method is expected to produce high quality RNA. The effect of precipitation solvents which have a low dielectric constant makes nucleic acids less hydrophilic and precipitates. The good quality of RNA can be seen with the high intensity of the DNA band produced and the low stain intensity. From the research, pure RNA was obtained in the isolation process with acetone-methanol (1: 1) precipitation solvent with an absorbance value of 1.81. So, from this study it was reported that the acetone-methanol (1: 1) precipitation solvent was the best solvent for precipitation. (*)
Author : Teguh Hari Sucipto
translator : Ausie