In this study, mRNA was isolated from mouse liver tissue using phenol-chloroform RNA extraction. The integrity of mRNA was tested by agarose gel electrophoresis and spectrophotometry (Nano Drop). Quantification of mRNA was obtained by (quantitative polymerase chain reaction) q-PCR. Based on mRNA quantification, the type of tissue was determined as obese mouse liver.
Having high quality, intact RNA is the first step in many basic molecular biology experiments, such as: nuclease protection assay, RT-PCR, RNA mapping. Quantity and quality of the starting RNA has a high impact on the accuracy of gene expression. In most of the RNA-based analyses the purity and integrity of RNA play important roles. In molecular biology and diagnostic applications the presence of high-quality intact RNA as a substrate would be suggested. (Raeymarkers, 1993). Practically, RNA is very sensitive to degradation. This might be happened via various reasons including: postmortem processes, sample handling or storage (Perez-Novo et al., 2005). Therefore, increasing interest was observed for the fast measurement of the quality of RNA using minor amounts of that. The major reason of that interest is that mRNA transcripts have become costly and sophisticated. The quantity and quality of purified RNA is not the same for different extraction experiments (Bustin et al., 2005). The procedure of extraction and purification must meet some standard criteria (Bustin and Nolan, 2004). An optimized RNA yield is achieved from tissue that has been divided into fragments before being frozen in liquid nitrogen. Most of the times conventional methods are not sensitive, and they are not specific for single stranded RNA (Imbeaud et al., 2005). Various methods were employed to assess the RNA integrity such as: Modern OD assessment using Nano-Drop, Classic gel OD measurement, Old fashioned denaturating agarose gel-electrophoresis (Agilent Technologies, USA) and Experion (Bio-Rad Laboratories, USA). There are some new spectrometric methods, such as NanoDrop (ND-3300, NanoDrop Technologies, USA) combined with RNA RiboGreen dye (Molecular Probes, Invitrogen, USA) is applicable as a tool for UV/VIS spectrophotometery in the case of ultra sensitive quantification of RNA. This system is able to work with a small quantity of sample which is the major advantage of this system and it would be dramatically important when samples are something like human biopsy or laser dissected. The polymerase chain reaction (PCR) is an accurate method because of because of repetitive multiplication of corresponding molecules. This can be a big disadvantage in quantitative measurement since small differences in multiplication factor cause significant differences in the quantity of product. In order to overcome this problem two methods have been introduced including: kinetic methods and coamplification methods. Amplification factor is being determined or compared in kinetic methods.
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In quantitative qPCR, either specific or non-specific identification using chemical methods provides the quantity measurment of the amplified product. There is a direct relationship between the detected amount at a certain point of the run and the initial amount of target in the sample. Gene expression analysis, pathogen detection/quantification and micro-RNA quantification are the applications quantitative PCR. For instance, the result of various treatment methods can be assessed on the level of mRNA transcription.
All chemicals and kits were obtained from Dr. Slitt's lab and followed by protocol (Slitt A, 2010).
RNA isolation of phenol-chloroform
Before starting, the pipettes and gloves should be clean with EtOH. During the whole experiment RNA contamination is the major concern. The procedure contains different steps such as: homogenization, phase separation, RNA precipitation, RNA wash, and re-dissolving the RNA. Tissue sample (50-100 mg) was extracted and homogenized in TRIZOL® reagent (1 mL). Homogenized sample was incubated at 15-30 oC for 5 min. The volume of chloroform (0.2 mL) was added to TRIZOL® reagent (1mL). Tubes were shaking vigorously for 15 sec and incubated at 15-30 oC for 2-3min. Samples were centrifuged at 12000g for 15 min at 2-8 oC temperature. Aqueous phase was transferred to a new tube and the RNA precipitated from the aqueous phase by mixing with isopropyl alcohol. Samples were incubated at 15-30 oC for 10 min and then centrifuged at 12000g for 15 min at 2-8 oC temperature. Supernatant was removed and the RNA pellet washed with 75% ethanol. The pellet let dried and RNA was dissolved in DEPC water.
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In the next step, in order to determine the RNA concentration, the absorbance was read using the nucleotide program at 260nm (UV) in duplicate. The sample was diluted to obtain RNA concentration of 1000.0 ng/μL. For agarose gel electrophoresis, loading buffer (15 μL) was added to 0.5 mL eppendorf tubes and diluted sample (5 μL) was placed. The sample was heated at 50-65 oC for 5 min and then ice used to cool it down. Sample was loaded on gel and then it run at 100 V (large gel) and 40-45 V (small gel) for 1-2 hr. The RNA integrity was checked after 20-25 min. For cDNA synthesis, RNA (1.2 μL) was dissolved in DEPC water (10.8 μL). The mixture was incubated at 65 oC temperature for 10 min time. The master-mix was prepared in eppendorf tubes including: buffer, inhibitor, dNTP's, and transcriptase. A volume of Master mix (7 μL) was added to each PCR tube and then the thermocycler was run for 1hr at 50 oC, and 5min incubation at 85 oC, and keeping at 4 oC.
For qPCR analysis, the cDNA was diluted with water. The master-mix was prepared including: buffer, water, and cDNA template. Master-mix (8 μL) was loaded on each well in 96-well plate, and 2 μL of primer mix was added to corresponding wells. Plate spin in centrifuge at 1500 rpm for 1-2 min and the plate loaded in Light-cycler 480 and new template created.
Result and Discussion:
Agarose gel electrophoresis
The agarose gel electrophoresis result is presented in Figure 1, shows the RNA was intact and obese.
Figure 1: Agarose gel electrophoresis
Basically, the integrity of mRNA tests by Aagarose gel. It showed two bands at 18s and 28s. The band intensity (Figure1) in upper band 28s is about doubled as lower in 18s. It indicated that mRNA has not been contaminated and the purity is acceptable. Spectrophotometry (Nano-Drop) was used to analyze the integrity of mRNA. The concentration adjusted to1000.0 ng/μL and the absorbance was recorded as 27.039 and 13.809 for two different wavelengths: 260 nm and 280 nm respectively. The absorbance ratio of 260 nm/280 nm which is 1.96 confirms that the integrity of isolated mNRA is qualified for further experiment.
Figure2. The data obtained via Nano-Drop
Averages of Cp values of samples were obtained using qPCR. To determine what is the type of tissue sources (lean or obese liver), MRP4 (Abcc4) was set as the target gene because in obese liver, the expression of this gene was in a higher level than the expression in lean liver (Slitt, et al., 2008). Housekeeping gene expression was measured as a standard. Average of MPR4 (Abcc4) gene had a Cp value of 26.94 for targeted gene and value of 15.565for housekeeping gene. These numbers demonstrate sample MPR4 gene take 26.94 PCR amplify cycles to reach the threshold point. Cp value results of different groups showed that some of samples have high level of MRP4 mNRA level while others are relatively low. The normalized value is shown in Figure 3.
Figure3. Average CP values for lean versus obese
The result showed sample K313 is extracted from obese liver based on the MRP4 (Abcc4) mRNA expression level. Due to the high expression of mRNA in K313, it takes less cycle to reach threshold point in q-PCR but for lean liver sample take more cycle to get high level of MRP4 mRNA expression.