[AWC 2nd Place] Unveiling genomic revolution: empowering quality of life through genetic engineering

Abstract

A stress response which is characterized by a permanent growth arrest and robust secretion of a

large number of biologically active molecules, is present in advanced plaques. The expression of

p16Ink4a, p53, and p21 as well as elevated senescence-associated -galactosidase (SA -Gal)

activity are senescence markers (Khemais, Toti and Schini-Kerth, 2017). The impact of

senescent cells on atherogenesis, however, is unclear. According to some studies, human and

mouse polymorphisms reduce the expression of p16Ink4a, which has since been connected to an

increased risk of atheromatous plaque formation. Following the review, a genomic tool has been

exploited for making a single cut into the p16(INK4a) gene in THP-1 macrophages.

Unexpectedly, it was found that senescent marker deletion could facilitate atherogenesis and

tumorigenesis, which was confirmed by the ability to see the effects of gene editing..

Keywords: Senescence-associated secretory phenotype (SASP); plaques; THP-1 macrophages;

atherogenesis.

1.0 Introduction

The CRISPR-Cas system has evolved into a popular and throughput benchwork in the genomic editing of plants, animals, and humans. Under the guise of genetic engineering, such a technology has also

enhanced the adaptability of crops. The transgenic tomato was approved by the Food and Drug Administration in 1994 (Kramer etal., 1994), and transgenic papaya was well-adapted to have high resistance to cyclic spot virus (Stirling et al.,2019). Such a genetic modification technology was

developed in 2013 to study the function of plant genes. Through a persistent improvement made upon this technology for the next 2 years, the supply of gene-edited crop germplasm increased. FDA approved CRISPR gene editing of waxy corn in 2016. Without using the stringent regulatory

process required for GM crops (Waltz et al.,2018), a mushroom that attenuated browning of skins (Agaricus bisporus) (Waltz et al.,2016) were introduced. Humans have gained benefits using

scientifically based technology to explore potential improvements. Discovering the ability of a small piece of chimeric single guide RNA (sgRNA) to pinpoint the essential Cas endonuclease to the precise location in the genome is what attracts so much attention in genetic engineering-based scientific research. The Cas enzyme cleaves the bound DNA at the target site to produce a double strand break (DSB), which is then repaired by the cells using either the non-homologous end joining (NHEJ) or homology directed repair (HDR) pathways. If the repair template is missing, the DSB is repaired using the error-prone NHEJ pathway, which causes frameshift mutations in protein-coding genes by adding or

removing nucleotides at the cut site in a pseudo-random manner. The high fidelity HDR pathway, on the other hand, is useful for repairing the DSB if the donor template with the desired DNA changes is present.The target locus of a gene typically uses a plasmid as the template to carry a lengthy sequence (Liu et al., 2019). Without a protospacer adjacent motif (PAM) at the target site, the Cas-guide RNA complex is unable to induce a separation of DNA. PAM works to limit the genomic regions that only

a specific Cas nuclease can cleave because different bacterial species have different PAM sequences. Cas 9 enzyme, for instance, can identify a PAM sequence of NGG, whereas Cas12a enzyme searches for a PAM sequence of TTN (Collias and Beisel, 2021). The Streptococcus pyogenes Cas9 enzyme has been reported to have better cutting efficiency, leaving blunt end cuts on both strands of DNA to undergo mutation when the strands repair themselves in contrast to Cas12, which leaves a 5' overhang to hinder

the accomplishment of certain edits. Additionally, the majority of mammaliancells are G-C rich, showing greater flexibility in locating the necessary GG for Cas9 targeting to a particular region of the gene. (Genomics Research from Technology Networks, n.d.) p16INK4a, is encoded by a gene found on chromosome 9p21, specifically at the INK4a/ARF locus. p16INK4a can inhibit cellular proliferation and is involved in the retinoblastoma protein (Rb). p16INK4a regulates development of a cell by halting the synthesis phase. When p16INK4a binds to CDK4/6 it prevents the formation of the cyclin D-CDK4/6 complex and the phosphorylation of the Rb family by CDK4/6. The Rb family members are kept

hypophosphorylated, which encourages binding to E2F1 and causes arrests in the G1cell cycle. Furthermore, cellular senescence, which is triggered by stress or aging, is also significantly influenced by p16INK4a. Senescent cells have a flattened and enlarged appearance (Romagosa et al.,2011), as well as senescence-related B-galactosidase expression and heterochromatic foci. According to research, p16Ink4a expression accelerates the aging process in mouse tissues, human skin, and kidney tissues, indicating that this protein is an important tumor suppressor (Romagosaet al., 2011). Some have also found that vascular cell senescence correlates with the pathogenesis of human atherosclerosis. Mammalian cells generally contain many p16INK4a genes, and THP-1 macrophages are not an exception. THP-1 macrophage is a human leukemia monocytic cell line that is actively used in the investigation of a wide range of mechanisms, cell-cell interactions, and pharmacology pathways (Chanput, Mes,and Wichers, 2014). Senescence caused the interaction between vascular cells and

macrophages, which led to atherogenesis. This scenario is best explained by the reduced production of nitric oxide by senescent vascular cells, which encourages the senescence of bone marrow-derived

circulating endothelial progenitor cells (EPCs), resulting in slow vascular healing (Minamino and Komuro, 2007). Relevant to CRISPR-Cas9 plasmid transfection, foreign DNA enters the respective cells which we have discussed. However, since the plasmids carrying gene of inerest in this engineering technology are

always large that approximately spans over 9 to 19 kilobases that will often implicate low transfection efficiency, and thus subsequent selection and purification are prerequisites and become necessary. In the transfection method, vector size shouldn’t be negligible. A promising approach that was done by taking the analysis confirmed by the researchers, the low transfection efficiency could be resolved via a standard electroporation, which further increases the physical contact among the target cells and

the engineered plasmid. Previously, a result had demonstrated that a transfected 15kb CRISPR-Cas9 GFP vector into non-small cell lung carcinoma (A540) had an inefficient transfection (4.2%) and prominently high cellular apoptosis which spanned around 91%. In contrast to using small transfected vectors into the cells, the transfection efficiency percentage increased drastically from 4.2% to 40% which had shown about 10-folds change, and cellular death decreased from 91% to 45% respectively (Søndergaard et al., 2020). Based on the result made, a hypothesis that could be formulated was that vectors as small as possible are capable of inducing better transfection efficiency. Profoundly, smaller size of vectors will have better transfection. There is an approved protocol which is suggested in the transfection method, including the proper kind of chemical transfection agent that is used in such a step. In our third practical, GeneJuice which is non-lipid based, was opted as the transfection agent in Opti-MEM medium which offers a stable environment for efficient transfer of foreign DNA or plasmid to enter the target cells, and is also suitable for high-throughput transfection in multi-well media,in which the media are compatible to what were selected in our third practical. In practical 4 regarding the harvest of genomic DNA from the transfected macrophages, TRIzolTM reagent will be used for a specific purpose. As such, the reagent permits the isolation of RNA, DNA which is concerned with our practical use, and protein from the samples, as well as offers lysis capabilities, no matter how difficult the samples are going to be lysed with respect to harvesting genomic DNA (www.fishersci.com, n.d.). Verification of genomic edits is unarguably important because of its principal aim, which is to verify the mutation induced by the gRNA targeting. This engages in PCR mix which contains forward and reverse Primer, high fidelity master mix, and also suitable template DNA with H20. The procedure of this verification is conformed to what we’ve learnt in polymerase chain reaction and agarose gel electrophoresis for the visualization of DNA fragments. For characterizing the mutation in the genomic

edits via those steps being mentioned, the DNA sequences are going to be observed to visualize if there is certain mutation existed, of the sequences which include insertion or deletion (INDEL), restriction fragment length polymorphism (RFLP) consisting of single nucleotide polymorphism (SNPs) combined with insertion or deletion (horizondiscovery.com, n.d.). Regarding the polymerase chain reaction and the gel electrophoresis, the Western blotting and also the fluorescent assay are also utilized in order to perform an optimal result of the DNA sequence visualization.

2.0 Methodology

Guide RNA designation via Brenching software

Two guide RNAs (gRNAs) were created using Benchling software based on the genetic sequence of the p16INK4a exon 1 gene and its PAM sequence before the actual CRISPR genome editing work in the animal tissue culture laboratory began. By selecting and importing the DNA sequence, the create

button was used. After selecting the"External database" tab, the search was conducted using the gene of interest"CDKN2A". GRCh 38 (hg38, homosapiens) was selected from the drop-down menu and imported. The CRISPR icon on the right tab was clicked for the selection of design and analysis of guides after the CDKN2A genome region had loaded on the screen. The finish button was clicked after

selecting the single guide. The tab was scrolled down on the sequence map, which had CDKN2A in the middle panel, to look for exon 1. Clicking on the button created Exon 1. Then, "Find genome matches" was selected after clicking the blue text "No region set" that was displayed. Set Genome Region was clicked after the option with the most matches was chosen. A list of guides with varying On-target scores, which indicate the efficiency, and Off-target scores, which indicate the specificity, were

displayed on the right panel. For a list of gRNAs overview, click On- and Off- target scores. The forward primer for the gRNA 1 had a 24 base pair design and targeted the sense strand of the gene sequence: 5'-CACCGTGGCCAGCCAGTCAGCCGA-3'.The reverse primer targeted the antisense

strand of the gene sequence and had a 24 base pair sequence: 3'CCGACGACGACGAGGTGCCGCCAAA-5'. The forward primer for gRNA 2 had a 25

base pair construction: Specifically targeting the sense strand of the gene sequence,

5'-CACCGCCCAACGCACCGAATAGTTA-3'. The reverse primer was created with a 25

base pair sequence that targets the antisense of the gene: 5'-CGGGTTGCGTGGCTTATCAATCAAA-3'.

CRISPR vector digestion and purification

The PX459 plasmid vector, Bbs I enzyme,10x PCR buffer, distilled water, and gRNA inserts were all prepared beforehand. First, the PX459 vector was digested by combining 4.1 L of plasmid vector with 1.0L Bbs I enzyme, 2.0 L 10x buffer, and 12.9L distilled water for an hour at 37°C. After incubation, the dephosphorylated digested plasmid vector was run on a 1% agarose gel at 80V for 30 minutes. Following band visualisation, the vector representing the band on the gel was cut using a “Promega”

kit for purification purposes. A NanoDrop spectrophotometer was then used to measure the concentration of double-stranded DNA. The linearized vector was kept in the ice box and used right away when necessary.

Short oligo insert annealing (gRNA) of gRNA

They were then added to a mixture for annealing and phosphorylation that contained 1 L of 10x T4 ligation buffer, 1 L of T4 polynucleotide kinase (PNK), 6 L of distilled water, and 1 L each of short sense

oligo inserts and short antisense oligo inserts. The mixture was then added to a thermocycler that was set for 30 minutes at 37°C. The temperature was raised to 95°C for five minutes before falling to 25°C at a rate of five degrees per minute.

Ligation mix and transformation of gRNA into competent cells

For each individual gRNA insert, the ligation mix was prepared using the materials listed below, which were as follows. As a result, there were 50ng X459 vectorS consisting of positive control with

undigested 2uL of PX459, negative control, gRNA1 and gRNA2 with BbsI-digested 4uL PX459 vectors. In regard to dilution of annealed oligos (gRNA1 and gRNA2), the dilution factor was evaluated in a ratio of 1:10, accompanied by addition of T4 DNA ligation buffer with 1uL of DNA ligase, as well as important dH20. Each ligation mix remained at room temperature for 1 hour and was subsequently added into the competent macrophages and incubated on ice for another 15 minutes. The bacterial cells were then subjected to a heat shock treatment at 42 degrees Celcius for about 1 minute, then being cultured on particular agar plates, followed by incubation overnight at 37 degrees Celsius.

Transfection of CRISPR/Cas9 plasmids

2.5ml (2 x 105 cells/ml) THP monocytes were seeded in a 6-well plate (5 x 105 cells/well) and THP1 differentiation was induced with 100ng/ml PMA for 48 hours. The culture medium was changed to 2ml of

Opti-MEM after two days. PX459, gRNA1-PX459, and gRNA2-PX459 were each added to a DNA mixture with 2 g. A 37oC pre-warming was applied to the opti-MEM. In each tube, 6 L of GeneJuice was mixed with 200 L of optiMEM. After that, the mixture was incubated for 15 minutes at room temperature. Following incubation, the mixture was gently mixed with the THP-1 macrophages before being

incubated for another 2 days in a 5% CO2 incubator.

Harvesting genomic DNA from potential knockout cells

The cells were harvested after Promega’s purification and post-transfection for 2 days. The culture medium was aspirated and the cells were scraped off with 1ml PBS. The cells were introduced to centrifugation at 1600 xg for 1 minute for recovery and the supernatant was discarded. 600 μL of Nuclei Lysis Solution and 3 μL RNAase Solution were added to the cell pellets and mixed well by pipetting, then sent for incubation at 37oC for 15 minutes. After allowing each of the mixes to cool to room temperature, 200μL Protein Precipitation Solution was added to each mix and allowed to cool on ice for 5 minutes. Following another round of centrifugation, the supernatants were transferred to a fresh tube and added with 600 μL isopropanol. After another round of centrifugation, the supernatant was removed

and 600 μL 70% ethanol was added. Once the final round of centrifugation was done, the pellet was allowed to dry. 50 μL DNA RehydrationSolution was then added to each pellet collected and incubated at 25 degrees Celsius for 10 minutes. The purified DNA for each recombinant plasmid and its

replicate were subjected to spectrophotometric quantification to determine the A260/280 reading and its corresponding dsDNA concentration reading.

Verification of gene editing

The extracted recombinant DNA plasmids from previous experiments were added to fresh tubes and the PCR mix consisting of the following contents were added to each recombinant plasmid reaction which was tabulated in the tables.

3.0 Result

Analysis of DNA content via agarose gel electrophoresis

The PX459 plasmid was digested by mixing it with Bbs I enzyme, buffer, and distilled water,

then the product was run through agarose gel electrophoresis to visualize the band. The ladder

was represented by lane 1 in Figure 1 below, while lanes 2 through 5 were the bands made up of

the digested plasmid PX459 by 4 groups. Sample 4 had a thicker band than that of other samples.

Plasmid DNA concentrations were also determined by using spectrophotometer, such values

were tabulated simultaneously.

Conduction of agarose gel electrophoresis of plasmid digested vector. Sample 4, as labeled by

the circled part, had a thicker band than that of other samples, implying the DNA

conenteannealed gRNA-1 should have bigger DNA content.

Absorbance and concentration of DNA through spectrophotometry

Table 2 indicated different concentrations and absorbances of digested plasmid PX459, annealed

gRNA 1 and annealed gRNA 2.

Culturing transformed macrophages that have received gene of interest in agar plates

Following the ligation of gRNA 1 and gRNA 2 with the PX459 plasmid vector as well as the transformation into DH5-alpha type of the competent macrophages, the bacterial cultures were settled upon LN/Amp agar plates labelled with positive control, negative control, accompanied by incubation of guided RNA for overnight. As illustrated, from figure 2 to figure 5, the number of colonies were observed following the incubation.

Figure 2 showed arrowheads which were represented as several single and scattered colonies found in the positive control of agar plates whereas figure 3 did not show any colonies present in the negative control of agar plates, same as whatever in figure 4 (annealed gRNA-1) and figure 5 (Annealed gRNA-2). This was found to be contradictory as the colonies were all supposedly to be present in figure 4, and 5 as well. In figure 2, there were approximately 80 colonies captured during the transformation process in the respective agar plates. Unfortunately, the agar plates that were re-done had not been taken photos,

generating repercussions as resulted above. As suggested, figure 4 and figure 5 should exhibit more colonies being cultured than that of positive control.

Visualization of absorbances at different wavelengths

After transfection and 48 hours of incubation, the DNA was extracted from the transfected THP-1 macrophages. The DNA was then measured using spectrophotometric analysis and seen on agarose gel

electrophoresis. Figure 6: The dsDNA concentration of genomic DNA extracted from THP-1 macrophages transfected with the PX-459 control vector, gRNA 1-PX459, and gRNA2-PX459 vectors was calculated. Also noted was the absorbance at 260 and 280, respectively. A positive control made up of SPL1 and SPL8 plates is shown in (B). (C) GRNA1-containing plasmid in SPL2 and SPL9 plates. D: Plasmid in SPL3 and SPL10 plates with gRNA2.

Agarose gel electrophoresis of transfected THP-1 macrophages

Figure 5 (Left-sided illustration): Visualization of agarose gel electrophoresis from left to right in which the first lane was the 100 base pair DNA ladder. Second lane indicated the negative control which was not transfected with the PX-459 vector. Third lane illustrated the positive control transfected with the PX-459 plasmid vector. Fourth lane demonstrated gRNA 1-PX459. Fifth lane was loaded with gRNA 2-PX459.

Unclear bands were seen from Lane 2 to Lane 5.Figure 6 (Right-sided illustration): Simulation data on agarose gel electrophoresis displaying PCR amplification products of genomic DNA isolated from the transfected THP-1 macrophages. From left to right, the content of the visual gel bands was as follows: 100 base pair DNA ladder, PX459 plasmid-transfected positive control, gRNAs1-PX459 and 2-PX459, negative control from THP-1 macrophages, and positive control. The unaltered wild type p16INK4a

exon 1 sequence was visible in the samples transfected with PX459 in bands of about 400 base pairs. p16INK4a exon 1 was successfully partially knocked out after gene editing in the samples taken from PC-3 cells transfected with gRNA-PX459 plasmids.

4.0 Discussion

A low positive value of DNA concentration interfering transformation efficiency percentage to be lower can be caused by low number of transformed plasmids entry, the colony of bacteria that had the plasmid was discarded unintentionally. For the agarose gel electrophoresis in Figure 1, only a single band was seen in each land. This was because the PX459 plasmid had been completely digested into its linearised. These bands were 300 base pairs in size or less. Also noticed in the PX459 lane were ghost bands. form, indicating successful digestion of plasmid (www.goldbio.com, n.d.). Small amount of DNA present in the plasmid caused the band to be thinner since the concentration of the plasmid vector recorded after passing through the spectrophotometry was relatively low. (www.labxchange.org,n.d.). Purity of the plasmid DNA and annealed gRNAs should be determined to prevent unwanted contaminants which would interfere with the following protocols. According to Table 1, the absorbance ratio at 260 and 280 (A260/280) for digested plasmid vector, gRNA 1 and gRNA 2 were 1.720, 1.253, and 1.769 respectively. Digested PX459 plasmid, gRNA 1 and gRNA 2 absorbed UV radiation at 260nm.

The absorbance at this wavelength was often compared with absorbance at wavelength 280 nm to estimate sample purity. A260/280 with value 1.8 For Figure caption: please indicate the below as Figure 1, followed by (a), (b), and so on. And briefly mention how the cloning and transformation were performed. Each figure should be an independent representation. The delineated description of the results had indicated pure DNA while a ratio of 2.0 indicated pure RNA (Lite, 2012). For plasmid DNA, absorbance value at 1.72 was slightly lower than 1.8, further showing the presence of a little but not impactful contaminants like proteins or phenol molecules. However, for the absorbance values of gRNA 1 and 2, the values were 1.253 and 1.769 which spanned around the normal purity range. This outcome represented a high amount of desired DNA inhabiting the gRNAs, which was most probably contributed by accurate volume of the annealing and phosphorylation mix. The contents of the whole mix were altered, then consequently influenced the purity of the gRNAs. Identification of specific contaminants could be explored by utilizing different ratios of absorbance to investigate whether these contaminants really manifested a huge effect in the following steps, especially the ligation and transformation part. For the result fromagar plates, there was a lot of white colonies formed on the positive control plate, and

merely 1 to 2 colonies in negative control, gRNA 1 and gRNA 2 plates. The colonies formed on positive control plate was deemed to be a correct result because the bacterial cells transformed with undigested plasmid will not be able to exhibit ampicillin antibiotic resistance, indicating the complete presence of the PX459 vector and no cleavage has occurred. According to Figure 3, there was no colony observed in the negative control plate since the reason behind was opposite to the positive control, which meant the PX459 plasmid was already fully digested. Moving on to gRNA 1 and gRNA 2 plates, supposedly, it should have approximately 10 colonies formed on each plate but according to Figure 4 and 5,no colony was seen. This result was not aligned to what was expected as DH5α cells have high transformation efficiency of around x10 9 cfu/μg so it is quite impossible to not have any colony if all the steps in protocol were followed strictly (Kostylev et al., 2015). So, this might be due to the undiluted annealed oligos being ligated with the plasmid and transformed into the competent cells. The gRNAs should be

diluted by 10 times before reaching room temperature to avoid inefficient ligation and melting of oligos. When the concentration of annealed oligos was too high, they would reanneal back and reject the ligation process with the plasmid, causing less or no transformants (Protocol for Annealing

Oligonucleotides, n.d.) The best molar ratio of plasmid vector to the insert should be 1:3 but somehow could be altered to 1:5, lying within the range from 1:1 to 1:10 (international.neb.com, n.d.). Another

possible reason led to failure of transformation was the exposure to an excessive number of freeze-thaw cycles. As the age and prior handling of the DH5α competent cells were unknown, it was possible that these competent cells may had been subcultured for other experiments, where it was thawed and frozen to be put back into storage before it was thawed again for the conductance of this experiment. If such an event had occurred, drastic changes in transformation efficiency may be observed, with nearly two-fold reduction in transformation efficiency being observed on some occasions. As such, using cells that

were not previously thawed may increase transformation efficiency (2020.igem.org,

n.d.). Not only that, the current transformation protocol applying heat shock method was not the most appropriate version for DH5α competent cells. In 1983, Douglass Hanahan proposed a new method

that enhance the competency levels of many E.coli strains but it was more complex to be

conducted. Hence, it was suggested to use the revised and modified Hanahan transformation method based on different conditions and requirements of the experiments in resolving the low transformation efficiency issue. (Chan et al., 2013) Cloning technique was deemed popular in biotechnology research but it had a fatal drawback in regards to its extreme time consumption. In this practical, it took

up almost 7 hours to complete the whole process. There were lots of waiting time and everyone should keep an eye on the timing to avoid over incubation as mammalian cells were very sensitive and easily damaged. Once a step went wrong, the whole practical was considered to fail since the results obtained the day later would be illogical to be discussed. Using agarose gel electrophoresis, the results of the verification of mutation in genomic edits can be seen after PCR is carried out. For the gel electrophoresis result, the first lane was a 100 base pair DNA ladder, the second was a negative control without a DNA template, the third was a plasmid vector alone, the fourth and fifth were plasmid with gRNA1 and gRNA2, respectively. The transfection of the plasmid was done incorrectly, resulting in negative DNA concentrations, which should not have produced the pale bands. Nevertheless, all lanes showed faintly visible bands, albeit ones that weren't very clear. Because even no DNA concentration

was left, the bands depicted on the result were not consistent with the idea of band thickness in gel electrophoresis. When the band is seen, it typically means there were some contaminants present, as Tomas suggested (ECHEMI, n.d.). This is because of the fact that negative controls are always

present. Contrary to the simulator's findings, which indicated that the negative control should not have produced a band, the presence of distinct bands on the gRNA1-PX459 and gRNA2-PX459 and the

positive control, respectively, confirmed that the negative control was not supposed to produce a band. The simulated results displayed very distinct, thick bands in the first row of each lane, which was related to the significant amount of DNA containing those specific base pairs (www.bioinformatics.nl, n.d.). Additionally, the thicker bands seen were 400 bp-long exon 1 fragments of the unmutated type. The

bands displayed, however, were nearly the same thickness in the specified row, making it impossible to determine which lane was more loaded with DNA from the simulated result regarding the row containing 400bp fragments. The positive control transfected with plasmid PX-459 vector, gRNA1-PX459 vector, and gRNA2-embedded plasmid vector had also indicated the band after the thicker one, but existed in thinner appearance, which was in fact a deletion mutation happening in exon allele of

p16INK4A. Based on the accurate simulator results, it is possible to predict the presence of the band using the characteristics of gel electrophoresis, which were found to be strongly correlated with DNA concentration and DNA molecular weight in terms of kiloDalton, respectively. The assumption

that a single base mismatch can result in conformational changes in the double helix that cause differential migration of heteroduplexes and homoduplexes is typically the basis for mutation detection in any type of dsDNA by gel electrophoresis (Körkkö et al., 1998).

5.0 Conclusion

To conclude, the gRNA design, digestion and purification of CRISPR vector, and annealing of short oligo inserts were successfully done. However, there were mistakes conferred on the ligation and transformation stages, causing no colonies formed on the gRNA 1 and gRNA 2 plates. There were several precautionary steps to be noted for improving the following lab work. First of all, be mindful of the important parameters for instance the volume of solution, weight of the solute or materials,

temperature and time of incubation which would induce major fluctuations in the results to obtain precise and accurate results. In future research, advanced and up-to-date manuals like Hanahan protocol could be used as reference to compare with the traditional method for yielding more reliable results. The absorbance of spectrophotometry should not be just restricted to A260/280 but other ratios like

A260/230 could be utilised to identify the types of contaminants present in the sample. Apart from those aspects, mutation of senescent markers could feasibly elevate the risk of atherogenesis as well.

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