One of the most widely used methods to determine gene expression is messenger RNA (mRNA) analysis. Reverse transcriptase is frequently used by researchers to create cDNA, which is frequently used as a template in RT-PCR to detect and quantify the expression of gene products. The process through which a gene's information is utilised to produce a functional gene product is known as gene expression (proteins). Real-time PCR (RT-PCR), western blotting, and the polymerase chain reaction (PCR) are some of the most important methods utilized to accomplish the purpose. The study of gene expression entails the use of RT-PCR to determine the presence or absence as well as decrease or an increase of expression of the particular gene (Livak and Schmittgen, 2014, p. 402).
The Objective of Gene Expression Analysis
The objective is commonly attained by measuring the degree of abundance of the transcript associated with that specific gene. The process occurs under certain conditions and when quantifying the changes of expression one can tell the concentration of protein likely to be produced by expression of the gene. Further, it is crucial to note that RT-PCR is a technique also referred as quantitative RT-PCR which measure amplification of the copy of cDNA as the process occurs. It is possible for the research to determine the starting concentration of the DNA. RT-PCR contains fluorescent reporter molecules such as SYNR Green dye and TaqMan probe that help one to monitor accumulation of products of PCR (Livak and Schmittgen, 2014, p. 402).
The Process of SDS-PAGE
As the amount of the target amplicon increases, do does the quantity of fluorescence that emitted from the fluorophore. The next process when studying gene expression is SDS-PAGE, an electrophoresis method used for separating proteins based on their sizes and charge. Protein of known mass is run parallel or simultaneously with the unknown, and the RF and mass can be plotted. The step helps in identification of the unknown protein.
Western Blotting
Finally, Western blotting is a technique used to detect the presence of specific protein in the complex mixture obtained from SDS-PAGE (cell extract) (Livak and Schmittgen, 2014, p. 403). In particular, it involves transferring the protein from the gel to a solid support. The particular protein being tested is confirmed using appropriately matched antibodies. Therefore, the current research utilizes these methods aimed at identifying gene expression of two typical housekeeping genes that lead to the formation of Actin in Hela protein (a human epithelial cancer cell).
Experimental Procedures
The first step in the process included lysing the human epithelial cells from the sample leading to extraction and purification of RNA. It was important to ensure attainment of high quality and intact RNA. The ratio of RNA absorbance (260nm) and those of proteins (280nm) was used to determine purity of the nucleic acid obtained. The next step involved the synthesis of cDNA from the RNA through reverse transcription followed by amplification of the target genes. The objective was achieved using the RT-PCR that helped to confirm the presence of the cDNA as the process occurred using SYBR Green dye (Freeman et al., 2013, p. 238). Then, the PCR products obtained were put under agarose electrophoresis which allowed separation of the cDNA product formed. Once the presence of the gene for Actin in Hela protein was confirmed, the next phase involved extraction of protein from the sample cells. The complex mixture of proteins attained was put under SDS-PAGE for separation based on their charge and sizes. The procedure involved running proteins of known sizes (standard) along the unknown in the sample. Finally, Western blotting was performed to determine the presence of the specific protein associated with expression of the two housekeeping genes (Freeman et al., 2013, p. 238).
Results
After extraction and purification of RNA, the sample was prepared for gel running. 8µg of RNA were prepared in 10µl of MilliQ water. 10µl of 2x RNA loading buffer was added. The RNA sample was denatured at 65°C for 10 minutes and the gel number, each lane was recorded as well. Electrophoresis was allowed to occur under 120v until bromophenol blue run about halfway. The procedure confirmed the presence of RNA.
Purity of the RNA Sample
As identified earlier, the concentration of the RNA used is 8µg of RNA in 10µl of MilliQ water. To determine the purity of the RNA sample, it was imperative to carry out spectrophotometry which provides an absorbance value of 1.926. If the value of absorbance obtained falls between 1.8 and 2.2, the sample of RNA is commonly considered to be pure and of high quality. At the same time, it is fundamental to note that three microliters of RNA was loaded onto the gel and three microliters of loading dye were used to perform the procedure. The process confirmed the presence of RNA in the sample. This can be seen in figure 1 located in appendices section.
Synthesis of cDNA
The next step involved the synthesis of the cDNA from the RNA template obtained from the above procedure. The result for in figure 2 in the appendices indicates the presence of cDNA in the RT-PCR sample obtained after amplification. The figure shows my cDNA dilution PCR gel that confirmed the presence of cDNA associated with the two housekeeping genes. The goal was achieved with 2µl of RNA was used in the cDNA reaction. In other words, this is the amount of RNA used in the cDNA reaction which resulted in the development of four bands from 6 to 10. The figure 3 (my actin cycle time PCR gel) in the appendices shows the results obtained by running a PCR amplification of target genes at different cycle times. Agarose electrophoresis of PCR products shows that PCR was successful and the resulting cDNAs were of correct size. The figure demonstrates the presence of cDNA for the two housekeeping genes that are of the correct size which indicates progression to carrying out western blotting. Finally, the figure 4 displays the results gained after Western Blotting procedure was performed. It is clear that there was detection of bands in the nitrocellulose paper. Reading from the standard protein ladder, actin in hela protein had a size of 70 kDa.
Discussion
One technical problem encountered during the experience regard the small volume of RNA collected from the sample. This was technical problems that may have influenced my conclusions about the expression of two housekeeping genes in the epithelial cells. More specifically, the challenge may have risen because or biological variations in individuals cells even though they were taken from culture under same conditions (Ginzinger, 2012, p. 503). However, it is critical to note that the result can be regarded as significantly reliable because the source of errors was minimized throughout the experiment particularly when it came to handling the RNA sample gained. Another critical factor that may have influenced the reliability of the result include the change in centrifugation temperature which could have altered the profile of gene expression profile (Whelan, Russell, and Whelan, 2013, p. 263). However, errors were minimized as the RNA was harvested from the cells as quickly as possible.The results obtained from the experiment indicate the two housekeeping genes are associated with expression of the hela protein. The findings are supported by an experiment conducted by Lucey, Nelson-Rees, and Hutchins (2009) who demonstrates that the genes are associated with epithelial carcinomas.For optimum success, it was imperative to consider the amount of dilution of cDNA. I realized that addition of too much or less cDNA affected the PCR cycles because of quick amplification of the materials. Therefore, it was crucial to determine the most suitable amount for the experiment. Too much dilution of cDNA often result in brighter bands on the gels, and extremely low quantities result in no amplification hence low-density images.The data on the cDNA dilution and cycle number vs. the PCR product communicate important information about merits of real-time PCR vs. PCR end points assays. It is evident that for RT-PCR it is imperative to dilute the cDNA sample, for instance, from 2 to 100 folds (Deprez et al., 2012, p. 67). The advantage of the process is that one can get an idea of the quantity to use by checking the intensity of end point PCR products. If it appears too faint, one should not dilute the same too much. In other words, the advantage of the RT-PCR is that any amount of cDNA can work as long as the quantity is not too much or less.I expected to get bands for help protein expressed by the two housekeeping genes from the western blotting procedure performed. From the figure 4, it is clear that there are no non-specific bands. If they were present, the best method of minimizing the problem is by using more specific probes and increasing the blocking time or adding reagents such as tween (0.1%) (Deprez et al., 2012, p. 67). The issue can also be solved by maintaining the recommended incubation time for primary and secondary antibodies.
References
Deprez, R.H.L., Fijnvandraat, A.C., Ruijter, J.M. and Moorman, A.F., 2012. Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depend on cDNA synthesis conditions. Analytical Biochemistry, 307(1), pp.63-69.
Freeman, W. M., Freeman, W.M., Walker, S.J., and Vrana, K.E., 2013. Quantitative RT-PCR: pitfalls and potential. Biotechniques, 26(1), pp.112-125
Ginzinger, D. G. 2012. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Experimental Hematology, 30(6), 503-512.
Livak, K.J., and Schmittgen, T.D., 2014. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods, 25(4), pp.402-408.
Lucey, B.P., Nelson-Rees, W.A. and Hutchins, G.M., 2009. Henrietta Lacks, HeLa cells, and cell culture contamination. Archives of pathology & laboratory medicine, 133(9), pp.1463-1467.
Whelan, J.A., Russell, N.B. and Whelan, M.A., 2013. A method for the absolute quantification of cDNA using real-time PCR. Journal of immunological methods, 278(1), pp.261-269.
