Dna And Rna Extraction Pdf
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And it is easy to induce lysis without any additive like mercaptoethanol. Lysis Buffer is composed high concentration of chaotropic salt. Carefully handle it.
innuPREP DNA/RNA Mini Kit
Advanced Techniques in Diagnostic Microbiology pp Cite as. Since thermostable Taq DNA polymerase was discovered in , nucleic acid amplification techniques have made great strides and contributed greatly to progress in the life sciences.
These techniques were introduced into the clinical laboratory and have produced great changes in diagnostic tools and tests. In particular, there have been many innovative molecular testing developments in the field of diagnostic microbiology. Culture methods of bacterial identification are labor-intensive and time-consuming.
However, they are simple, cheap, and remain the gold standard. It also is possible to perform antimicrobial susceptibility testing on cultured isolates, so conventional culture methods with biochemical phenotyping are still the most common procedures performed in clinical microbiology laboratories [ 1 ].
To further assist in microbial identification, nucleic acid amplification has been introduced in the clinical microbiology laboratory. Such testing was initially done for viruses, allowing detection of small amounts of viral nucleic acid quickly. Similar tests also have been applied to bacteria, especially those that require cell cultures, are difficult to grow on routine culture media, or are slow growing such as Chlamydia , Neisseria gonorrhoeae , and Mycobacterium.
In addition, there are ongoing attempts to apply these new techniques for routine clinical microbiology testing, including the diagnosis of sepsis [ 1 ].
The development of nucleic acid amplification has proceeded at an unprecedented pace and achieved higher sensitivity and specificity [ 2 ]. However, in order to obtain satisfying results with this new technique, the testing must go through several important steps. Preanalytical testing variables comprise sample collection and preparation, specimen transport and storage, stability of the nucleic acid in the samples, and nucleic acid extraction [ 3 , 4 ].
Nucleic acid extraction is the first step of any amplification experiment no matter what kind of amplification is used to detect a specific pathogen [ 1 , 5 ]. It is a crucial preanalytic step in the development and performance of any successful molecular diagnostic method and ensures a reliable result [ 3 , 4 ].
We must pay attention to the technical progress of the nucleic acid extraction as well as to the method for amplification and detection of nucleic acids in order to obtain satisfactory results. Nucleic acid extraction consists of three major processes: isolation, purification, and concentration. Commercial extraction kits are commonly used in the clinical microbiology laboratory [ 2 ].
These kits provide the essential requirements for nucleic acid extraction. These essential requirements have been well described by Boom et al. It is preferred that there be no requirements for specialized equipment or special knowledge and skills.
The final nucleic acid should be pure and easy to modify for various amplification techniques. The reagents and their product should be harmless, and the process of preparation should resist contamination with other specimens.
If the final volume of eluate is small, detection limits are maximized. When we deal with clinical specimens, we also should consider the elimination of potential inhibitors of the DNA polymerase and the removal of pathogenicity from hazardous pathogens as well as good target recovery and establishment of the integrity of nucleic acid targets [ 2 ]. Ideally, the final target is pure nucleic acid without amplification inhibitors or contaminants such as protein, carbohydrate, and other nucleic acids [ 8 ].
There are a few points to be specially considered when we consider the use of nucleic acid extraction in the field of clinical microbiology. The targets for nucleic acid extraction are diverse. They can be the cultured bacterial isolates themselves.
Alternatively, we can use culture media, including blood culture bottles or various clinical specimens such as sputum, stool, urine, tissue, or cerebrospinal fluid [ 1 ]. In terms of nucleic acid extraction, they may target the same nucleic acid, but they have different implications for the extraction procedure itself. Nucleic acid extraction from cultured bacteria is relatively simple because they are pure colonies and they contain large numbers of organisms.
However, we need to recognize that gram-positive bacteria have thick walls, such that nucleic acid extraction is more difficult than it is from gram-negative bacteria, which have thinner walls [ 5 ].
For clinical specimens, the details of the method depend on the characteristics of each specimen. It is important to remember that the subject is nucleic acid, not of humans, but of bacteria, virus, or fungus.
If we extract from clinical specimens containing human cells, we cannot help mixing human DNA and sometimes, recovery of microbial DNA can be diminished by the presence of human DNA. The basic principle of this method is to use the difference in density between the cesium ion and water and intercalation of EtBr, which shows good results for separation of various DNAs and the procurement of high-yield DNA [ 11 ].
However, it has important limitations in that it requires an expensive ultracentrifuge and considerable time, it is difficult to perform, and EtBr is harmful [ 7 , 8 ]. Consequently, this method is not suitable for clinical microbiology and has not been used in the clinical laboratory. Phenol—chloroform extraction is widely used. The process consists of vigorous mixing of phenol—chloroform solution and sample followed by centrifugation [ 7 ].
Phenol does not completely inhibit RNase activity, and this characteristic enables isolation of nucleic acid by combination with chloroform and alcohol [ 12 ]. After centrifugation, the upper aqueous phase containing the DNA can be separated from the lower organic phase containing denatured proteins, and DNA can be precipitated by adding ethanol or isopropanol with a high concentration of salt [ 8 ].
This method is also used for RNA extraction by concomitant use of guanidinium isothiocyanate. This combination can overcome the limitation of RNA extraction using the guanidinium isothiocyanate itself, so RNA could be isolated conveniently using a single-step technique by Chomczynski et al.
Total RNA is recovered by precipitation with isopropanol after separation of the upper phase containing the total RNA from the lower phase containing DNA and proteins [ 12 , 14 ]. Solid-phase extraction uses a spin column operated by centrifugal force allowing DNA to be purified rapidly and efficiently without the limitations of liquid extraction, including incomplete phase separation [ 8 ].
Solid-phase extraction using silica now is one of the most common methods for nucleic acid extraction. Silica that possesses a positive charge combines strongly with DNA, which possesses a negative charge, so it can enable rapid, pure, and quantitative purification [ 7 ].
In , Boom et al. The principle of this method is that it immobilizes DNA onto its particles in the presence of a chaotropic agent. It takes only a short time and can be applied to clinical specimens as well as to DNA and bacteria. The process of solid-phase extraction involves cell lysis, nucleic acid adsorption, washing, and elution [ 7 , 8 ]. Column conditioning is obtained using a buffer at a particular pH [ 19 ].
The nucleic acid will be released after cell lysis and decanting of lysis buffer into the column. Nucleic acid adsorption is completed in a chaotropic salt solution [ 19 ]. Washing buffer contains a competitive agent and can remove contaminants such as proteins and salts. In elution, TE buffer is applied to the column so that purified nucleic acid will be released [ 19 ]. Extraction principles using magnetic silica particles. Nucleic acid extraction from clinical specimens is quite different from that from cultured isolates of bacteria or fungi.
This extraction step can influence the subsequent performance of the diagnostic tests; the efficiency of nucleic acid extraction is related directly to the sensitivity of the final test results [ 21 ]. Each clinical specimen has diverse characteristics. Blood and stool are composed of many substances, and among these, heme and bile act as inhibitors of amplification and should be removed [ 5 ].
We can find the comparison results for nucleic acid extraction; however, this cannot ensure that we can adapt this result to different specimens and pathogens. In previous reports, herpes simplex virus DNA was isolated relatively easily from genital swabs [ 5 , 22 , 23 ], but obtaining bacterial DNA from stool samples was more complex [ 5 , 24 ].
To overcome these limitations, the extraction method must be evaluated before routine testing of specific pathogens from specific specimens.
For detection of clinically important viruses, extraction efficiency was evaluated in various specimens, including serum, urine, and cerebrospinal fluid, and good performance was confirmed [ 2 , 25 , 26 ]. However, we should not extrapolate these specific results to all types of virus and specimens. Tissue is an important clinical specimen for diagnosing localized cytomegalovirus infections in a transplanted organ as biopsy is a common method used to evaluate potential CMV infection [ 27 ].
However, tissue specimens have problems because the large amount of human tissue contains cellular DNA, proteins, and other materials [ 28 ]. So, a more complex step to extract the nucleic acid of the microbial pathogen is needed. Most commercial kits for tissue specimens extract human nucleic acid also. In recent years, we have been able to extract the viral nucleic acid from clinical specimens having cellular components, and there have been a few trials of these kits to detect various clinically important viruses [ 29 , 30 , 31 ].
There is one other report concerning the extraction of six viruses from clinical cellular specimens, and the investigators compared four commercial extraction methods [ 28 ]. The viruses included in this study are BK virus, cytomegalovirus, Epstein—Barr virus, human herpesvirus 8, herpes simplex virus, and varicella-zoster virus.
All four kits could extract DNA from all six viruses. Stool is an important clinical specimen for the detection of viruses causing diarrheal illnesses. The results can be affected by the efficiency of nucleic acid extraction from stool, because stool is a mixture of many unrecognized materials, including bacteria, protein, and other cellular materials. So, stool specimens are considered one of the most difficult specimens for nucleic acid extraction in the clinical laboratory.
In one report by Peiris et al. However, the detection rates were quite different in another report: only It is suggested that the difference in positive rates is a consequence of variations in the RNA extraction method [ 34 ]. Clinicians place great emphasis on the detection of bacteria and fungi in blood. Therefore, nucleic acid extraction from blood has become very important. Many researchers have found that there are numerous PCR inhibitors in blood culture bottles such as sodium polyanetholesulfonate SPS and hemins [ 35 ].
To reduce the detection time, the serum, plasma, or whole blood is used as a main specimen for detection of bacteria and fungi. Serum or plasma is more efficient and convenient than whole blood because whole blood includes many PCR inhibitors [ 37 ].
Most commercial kits showed a high recovery rate of pathogen DNA, but only those methods that used heat lysis with an alkali wash could remove PCR inhibitors.
Detection of brucellosis was highly sensitive even though Brucellae are facultative intracellular pathogens [ 38 ]. Similarly, kits containing proteinase K showed better yield of Brucella in serum specimens [ 39 ]. However, to enhance the sensitivity of PCR amplification, whole blood is considered as a final target because it contains more pathogens than serum or plasma [ 40 ]. Although the nucleic acid kits were not developed to extract microbial DNA from whole blood, all commercial kits are able to do so [ 21 ].
In recent years, we have been able to use several automated systems to extract bacterial or fungal DNA from whole blood [ 30 , 41 , 42 ], although they are expensive. They are suitable for high-throughput detection [ 43 ]. It also is important to consider the concentrations of pathogens.
However, the results were different when there were low concentrations of tachyzoites in blood [ 45 ] vs. We can see the similar result in Chlamydia pneumoniae detection from stools [ 34 ].
The positive rates were lowered when the RNA concentrations dropped and this confirms the clinical importance of the extraction methods used for stool samples.
Nucleic Acid Extraction Techniques
Metrics details. RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Extraction of high quality nucleic acids is difficult from neuronal cells and brain tissues as they are particularly rich in lipids. In addition, most common RNA extraction methods are phenol-based, resulting in RNA that may be incompatible with downstream applications such as gene expression. The accuracy of gene expression evaluation is influenced by the concentration and quality of input RNA. Starting with a low quality RNA may compromise the results of downstream applications which are often labour-intensive, time-consuming and very expensive [ 2 , 3 ]. To ensure acceptable total RNA quality, the RNA extraction procedure must fulfill a number of requirements: including, the final preparation must be free from protein, genomic DNA or enzyme inhibitors and must not include any phenol or alcohol carryover which may compromise downstream reactions [ 4 ].
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innuPREP DNA/RNA Mini Kit
These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years.
English Stay here. English US Stay here. German Stay here. English IN Stay here. The genomic DNA and total cellular RNA are available for subsequent downstream applications after only 15 to 40 minutes, each in their own reaction vessel. Sign up here.
Sign in Sign up. RNA Extraction. This article summarizes commonly used methods and kits for RNA extraction, presents the results of a survey conducted by Labome of randomly selected formal articles on RNA extraction and purification, and discusses recent comparison studies and two most popular extraction kits TRIzol and RNeasy. Cell lysis and dissolution Cell lysis can be achieved using buffers or reagents containing chaotropic agents such as guanidinium isothiocyanate, guanidinium chloride, sodium dodecyl sulphate SDS , sarcosyl, urea, phenol or chloroform. Alternatively, repeated organic extraction using phenol and chloroform, or dissolving the sample in buffers containing guanidinium salts, can also be used to remove proteins.
To standardize an RNA ribonucleic acid extraction protocol in children's saliva specimens.. Comparison of two RNA extraction methods was established; methods assessed concentration, quality and yield. Data were analyzed through measurements of central tendency and dispersion, frequencies and percentages. Analysis of cell amounts per saliva ml revealed a mean of ,
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