The technique used is PCR and samples was placed on agarose gel and examined under UV light. In figure 1, the ladder (L) on the first well is used a guideline to identify the approximate size of a molecule on a gel, using the principle that molecular weight corresponds to migration rate through a gel (Lee et al., 2012). Using the ladder from figure 2 to detect base pairs at each level, samples 1,2,3,4,5 and 6 can be seen at 500 base pairs from figure 1. This suggest that the molecular weight for HeLa cell in sample 1-3 and GAP-DH in sample 4-6 are identical. Having a constant molecular weight between the HeLa cells and GAP-DH ensures that protein loading is the same across the gel and allow evaluation of differing levels of expression between the samples with treated and untreated with ATO (Kozera and Rapacz, 2013).
Although the samples have the same molecular weight the band intensity varies. Between sample 1-3, the intensity of the bands gets lighter with sample 1 being the darkest band and sample 3 being the lightest. Band intensities are used to measure the mRNA levels of a gene when they are considerably different from the mRNA levels for the control gene (Bradford et al., 2005). According to Thermofisher.com, partially degraded RNA appears as smeared bands with increasing lower molecular weight. Therefore, the intensity of the band relates to the level of gene expression with the darker the band the higher gene expression and lighter the band the lower gene expression.
In figure 1, sample one has the darkest band as it contains the untreated HeLa cells and would have the highest level of gene expression as there was no ATO used to down regulate the E6 gene. Sample 2 shows a lighter band in comparison to sample 1 as 2.0 ?l of ATO was used on the HeLa cells which suggest that there is lower gene expression and ATO has down regulated E6. Sample 3 contains 5.0 ?l of ATO on the HeLa cells, this is a higher concentration of ATO added onto the HeLa cells compared to sample 2. In figure 1, the band shows a lighter intensity in comparison to sample 2 which suggest that there is a lower level of gene expression and E6 gene has been down regulated further with increasing concentration of ATO.
However, sample 4-6 contains the control GAP-DH which shows a consistent high intensity of the bands suggesting high level of gene expressions. Housekeeping gene are involved in cellular maintenance, therefore, constantly expressed in all cells and conditions (Eisenberg and Levanon, 2013). Also, the constant high level gene expressions from GAP-DH assures that ATO treated HeLa cells has been down regulated.
The last well contains NTC which appears with no bands. NTC detects contamination from external factor and monitors primer-dimer formation which may lead to false positive results (Bacich et al., 2011).This suggest that experiment was completed without interference from contamination and no false positive results were produced.