Dna-Rna Extraction, Lab Report Example

Abstract

Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA) are two essential coding molecules that direct life processes. Many laboratory experiments revolve around the understanding of these nucleotides. It is therefore necessary to determine a way that will enhance our ability to effectively extract these molecules from cells, which will demonstrate benefits in both the laboratory and the medical setting. In this experiment, an organic extraction using Trizol will be performed for RNA extraction and an extraction method using phenol-chloroform for DNA will be used. According to the nanodrop reading, the A260/A280 results were 1.97 for DNA sample and 2.15 for the RNA sample. This indicates that the sample did not become contaminated during the extraction process. Therefore, these extraction methods would be useful for additional molecular biology needs. In a laboratory setting, crime scene investigators can use this process to ensure that they would gain maximal yield from small amounts of human body fluids gathered from crime scenes. In a medical setting, physicians would be able to use this technology in order to better understand the genomic differences that exist in many types of cancers.

Key Words: DNA, RNA, extraction, yield, nanodrop, Trizol, quality, quantity, degredation

Introduction

Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA) are essential macromolecules because they are responsible for directing the reproduction of cells in terms of the central dogma theory (Andrew & Watson, 2003). Furthermore, DNA stores the genetic code of all living things, and it is minute differences in the identity of these structures that contribute to our characterization. Even though RNA is primarily responsible for converting DNA to mRNA during transcription, there are many other types of RNA available in the human body that have enzymatic activity and silencing activity (Pal et al., 2006). Many additional types of RNA are little understood in the scientific community and scientists believe that gaining a greater understanding of these molecules will contribute to a greater understanding of life as a whole.

The Human Genome Project has piloted the interest of scientists in genomic material, and it has allowed us to understand more about the genes contained within the human body and how these genes divulge for each individual. It allowed us to gain insight into human disease based on an increased understanding of the relationship between coding nucleotides and the protein it is ultimately responsible for producing (Olby, 1994). As a consequence of the knowledge gained during the Human Genome Project, there is an ever increasing desire for scientists to continue annotating both the human genome and genomes of plants or animals. The hope is that gaining a greater understanding of evolutionary relationships, or phylogeny, will help us understand more about human origins, which will ultimately help us determine new ways to create models for human disease.

In order to effectively study RNA and DNA, it is necessary to perform efficient extractions from cells. Conventional DNA extraction included exposing cells to cold ethanol, and the white silky substance would reveal itself. However, this results in the DNA become too delicate for many future experiments. Thus, it is necessary to add a variety of buffers that will allow the DNA to remain protected throughout this process. Although basic RNA extraction techniques have existed for several decades, a major issue in this field regards the difficulty it is to extract RNA in a stable manner. Since RNA is a single helix, it is particularly sensitive to heat, which causes it to be denatured rather rapidly. Therefore, extraction techniques should focus on the need to keep this macromolecule intact.

Since there is a need to perform high-quality DNA and RNA extractions, this project will focus on retrieving high quality, purity, and quantity of nucleotide samples using animal DNA. The protocols that were selected for this purpose claim to achieve high yield, and this hypothesis will be tested throughout this experiment.

Materials and Methods

A common DNA extraction protocol was utilized to isolate the DNA from animal gut cells. To prepare the cells for extraction, approximately 100 mg of cut contents were separated and disposed into a tube containing 300 mg of Zirconium beads. This solution was suspended in TN150 buffer to prepare the cells for lysis. After vortexing, the supernatant was discarded and the pellet was washed twice using the TN150 buffer. This was repeated several times to ensure that the unwanted cell contents would not contaminate the DNA sample. After the final wash, the pellet was resuspended in the TN150 buffer. It was then placed on ice for several minutes and then centrifuged. 500 microliters of supernatant was then removed to a microcentrifuge tube and the same concentration of TE buffer was added to it, and the two chemicals were then centrifuged together. After cleaning the cells using this process, chloroform-isoamyl alcohol was then added to the solution to perform the physical DNA extraction. The supernatant was removed and the process was repeated several times to ensure maximal DNA yield. After this process, one mL of ethanol was added in addition 50 microliters of 3M sodium acetate, completing the DNA extraction and storage process. When the DNA was required for use, it was centrifuged for 20 minutes at around 4 degrees Celsius. The precipitated DNA was then dried and stored in 30 microliters of TE buffer, pH 8.

The RNA was extracted using the Precellys method. To begin this part of the protocol, dry ice was prepared in the cooling machine, which was then calibrated according to a previously written protocol. The same amount of tissue was weighed for the DNA extraction process as was weighed for this section of the protocol. 100 mg of animal gut tissue was placed into a tube containing lysing beads and was left on dry ice. 1 mL of TRIzol was added to each tube before homogenizing. Samples were then incubated at room temperature for 20 minutes. All centrifuging steps took place at 4 degrees Celsius to protect against heat denaturation. After centrifuging the homogenized mixture, 200 microliters of chloroform were added to the mixture, which was then vortexed and incubated at room temperature for several minutes. The solution was then centrifuged again and transferred to a new tube and where 250 microliters of isopropanol, 250 microliters of high salt solution was added. The contents were vortexed and centrifuged. After pouring off the supernatant, the pellet was submerged in ethanol. The precipitated RNA was immediately dried on ice. It was then solubilized and the quantity was measured using the RN600 Nano Assay.

The RNA Screen Tape assay was then used to assess the quality of the extracted RNA. First, the reagents were placed at room temperature to ensure that the integrity of the RNA would be protected during this process. All reagents were vortex mixed before use for the same purpose and RNA samples were thawed on ice. 5 microliters of RNA sample buffer was then added for every 1 microliter of RNA sample concentration. This process was repeated for the RNA ladder control as well. The solutions were then vortexed thoroughly and centrifuged to position the RNA sample at the bottom of the tube. The sample was then denatured systematically, first heating the samples to 72 degrees Celsius for 3 minutes, then by placing them on ice for 2 minutes, and lastly collecting the samples. For sample analysis, the animal gut DNA samples were placed onto the 2200 Tape Station and the machine was set to run. The results were then observed and recorded for further analysis.

The Molecular Probes System by Invitrogen was the device used to measure the RNA. To do so, the Qubit Reagent was added to the Qubit Buffer to form the Qubit Working Solution. Different amounts of this solution were then added to the kit standards, which worked as a control, and to the gut animal RNA samples. After the solution was added to the tubes, they were vortexed thoroughly and incubated at room temperature before the findings were read on the Molecular Probes device and recorded for later analysis. The concentration of both the DNA sample and the RNA sample was examined using the nanodrop system. The machine was cleaned prior to use to prevent contamination, and standards were measured to ensure accuracy before the samples were placed on the machine. The results were then recorded for later analysis.

Results

The nanodrop system revealed that at an absorption of 260 wavelengths, the DNA measured 7.49 using a 374.66 ng/ul sample. At an absorption of 280 wavelengths, the DNA measured 3.802 using a 374.66 ng/ul sample. When the size of the sample nearly tripled at 939.46 ng/ul for the RNA reading, the measurements at both the 260 and 280 wavelength absorptions tripled as well, to 23.487 at an absorption of 260 and 10.937 at an absorption on 280. Thus, the nanodrop results can be considered reliable. The A260/A280 results were 1.97 for the first sample and 2.15 for the second sample, indicating that it is unlikely that these nucleotides have become contaminated with organic solutions during the isolation process. The total results for the nanodrop reading can be viewed in table 1. The Molecular Probes kit was used to measure the RNA only. The device gave back the reading that the concentration was 294 ng/ul, after the reading was adjusted for considering the 1:4 dilution that was performed.

Discussion

During the DNA and RNA extraction process, it is essential to emphasize the need to maximize the nucleotide concentration yield in order to ensure that it could be used for molecular biology applications. Ultimately, this requires precipitating the sample with ethanol several times to ensure that all usable DNA or RNA has been removed from the sample. Furthermore, when working RNA, there is a need to ensure that the sample remains on ice between steps. RNA exposure to heat can denature the macromolecule, rendering it useless for scientific study. Although care has to be taken when working with and storing DNA as well, degradation is less of a concern due to the degree of stability that is an intrinsic property. It is essential to consider however, that contamination of a DNA sample is more likely if proper care is not taken to cover ones hands. An example of this is contamination with human DNA or exposure of the experimental sample to proteases that will actively degrade the sample.

There are many molecular techniques that rely upon DNA extraction to ensure proper results. For example, the polymerase chain reaction (PCR) is used to multiply small stretches of DNA for both diagnostic and experimental use. If the DNA is contaminated, it will be impossible to determine whether the DNA of interest was replicated when it is run on a polyacrylamide gel. In addition, while PCR was created to allow for the replication of small amounts of DNA, less PCR cycles are needed if there is more product. Yet other applications require a high yield of DNA to ensure that the DNA extraction process does not need to be repeated frequently, which would hinder the progress of the project on the basis of time wasted to do unnecessary procedures.

An example of a situation in which it would be imperative to correctly extract DNA from a tissue sample is in crime scene investigation. Typically, crime scene investigators need to work with trace evidence, which is smaller amounts than one would normally work with in the laboratory setting. Therefore, if the police field team is only able to gather a microliter of blood from the crime scene, it is the responsibility of the forensic scientist to extract an optimal amount of DNA and RNA from the blood in order to perform PCR. The ultimate goal of this process is to identify the criminal’s genetic profile, which is only possible if enough DNA could be generated from the retrieved sample.

An additional application of DNA and RNA extraction is relevant to the field of oncology. Since cancer is an occasionally a genetic abnormality, scientists profile many patient samples in order to catalogue frequent genetic mutations for each different type of cancer (Bernstein et al., 2008). Therefore, when working with rare patient samples, it is necessary to gain the proper amount of DNA from the sample immediately. Even though these samples could be grown in vitro, it is essential to consider that any additional passaging that is done can alter the genetic sequence of the sample due to the heterogeneity of human tumors. This is an important consideration because it is often difficult to recruit human subjects to research protocols, and this process is made even more difficult when a rare cancer is being studied (Hoeijmaker 2009).

Since gut DNA and RNA was extracted in this particular example, it is useful to consider the ways that this extraction process could prove valuable for this particular cell type. Currently, there are many gut diseases and humans that are not well understood. Among these are Chron’s disease and ulcerative colitis, which very few doctors know enough information about to treat effectively in all cases. Furthermore, there is currently no cure for his painful disease. Therefore, the DNA and RNA extracted from guts can be used to gain a greater understanding of the genetic relationship between genes and the material that is transcribed. Since most specialized cells have genes that are turned on and off, a complete DNA and RNA profile can help achieve this understanding. Comparing this profile to healthy tissue may be able to provide insight into the genetic differences that individuals with this disease have compared to healthy individuals in addition to detailing the biological mechanisms that can be interfered with to return afflicted patients to normal.

Ultimately, science has come a long way since human’s first extracted DNA purely using rubbing alcohol. Although this was a necessary discovery in our history, we have since modified this process in order to ensure that we gain pure nucleotide samples in addition to an adequate concentration yield. This experiment demonstrated that the techniques discussed are effective, but it is important to consider alternative methods and compare their efficacy. It is essential that we remain unbiased in this process and continue to consider how we can improve the efficacy and accuracy of the scientific techniques that are valued by our laboratory and medical professionals. Future studies on DNA and RNA extraction techniques will therefore focus on optimizing the efficiency of this process in addition to minimizing the amount of time it will take.

Additional considerations that can be taken in future experiments is how to prevent against human error. Since the RNA and DNA extraction processes involve carefully following a protocol, there is room for error at several steps. Ways to compensate for this would be to build machines that can mechanize the process, which would reduce chances of error. However, these machines would need to be regularly stocked with buffer and cleaned to avoid contamination. It would be in the best interest of pharmaceutical and biotechnology countries to develop these machines so that they may optimize this process.

Conclusion

The DNA and RNA extraction techniques used in this experiment allow for effective extraction of nucleotides. It is essential to include the repeated isolation steps to ensure that the samples have the highest concentration of nucleotide available. This sample can then be diluted for future purposes. Furthermore, the nanodrop measurement techniques is an effective method to verify the concentration of nucleotide present in a given sample. Additional measurement techniques are not necessary in order to ensure that the proper concentration has been determined, although they can be utilized for additional certainty. While the Trizol extraction method was beneficial for RNA extraction and the phenol-chloroform extraction method was useful for the DNA, it is important to consider additional extraction techniques and to continue optimizing this process in order to ensure maximal benefit to the research and medical fields.

Reference

Bernstein H, Payne CM, Bernstein C, Garewal H, Dvorak K. (2008). Cancer and aging as consequences of un-repaired DNA damage. In: New Research on DNA Damages (Editors: Honoka Kimura and Aoi Suzuki) Nova Science Publishers, Inc., New York.

Berry, Andrew; Watson, James. (2003). DNA: the secret of life. New York: Alfred A. Knopf.

Hoeijmakers JH. (2009). DNA damage, aging, and cancer. N. Engl. J. Med., 361 (15):    1475–  85.

Olby, Robert C. (1994). The path to the double helix: the discovery of DNA. New York: Dover Publications.

Pál C, Papp B, Lercher M. (2006). An integrated view of protein evolution. Nature Reviews Genetics, 7 (5): 337–48.