Extraction of DNA has to be an efficient process. It is necessary to ensure that the DNA extracted is pure enough for subsequent analysis. DNA can be extracted from several samples. It is easy to extract DNA from some samples like blood. On the other hand, it can be very difficult to extract DNA from degraded tissues. However, unlike RNA, which degrades easily, DNA is more stable and as a result, DNA extraction is easier than RNA extraction. Once DNA has been extracted, it needs to be quantified so that an estimate of the DNA concentration can be made. This quantitation is important for subsequent analysis.
There are many methods available for extracting DNA. The choice of method depends on a number of factors, including the type of the sample, the quantity of the sample and the speed of extraction. Each factor dictates the method of DNA extraction. For example, if the sample is peripheral blood and an adequate amount of blood is available, DNA can be extracted using the phenol chloroform method. On the other hand, if the amount of sample is very small as in bloodstains, the method would differ. If possible, one should choose a method where the DNA extraction procedure can be automated. Success depends on extracting the maximum amount of DNA from a sample. At the same time, the PCR inhibitors should be removed from the extracted DNA since PCR inhibitors inhibit downstream applications. If possible, hazardous chemicals should not be used during the DNA extraction process. Finally, the experience of the laboratory staff is important; experienced technicians are aware of the nuances of the extraction process and the quality of the final product is much better.
<H2>General principles of DNA extraction
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There are three stages in the DNA extraction procedure. These are as follows:
Disruption of the cellular membranes, resulting in cell lysis
Separation of DNA from the denatured protein and other cellular components.
Some of the extraction methods commonly used are described next.
<H3>The Chelex method
Chelex is a resin copolymer containing paired iminodiacetate ions. It has a very high afï¬nity for polyvalent metal ions, such as magnesium; it, therefore, chelates and effectively removes them from solution.
The extraction procedure is very simple. The Chelex resin, which is supplied as beads, is made into a 5% suspension using distilled water. The tissue from which the DNA is to be extracted is incubated with the Chelex suspension at 56°C for up to 30 minutes. Proteinase K is added. The proteinase K digests most of the cellular proteins. This is followed by incubation for 8-10 minutes at 100°C to ensure that all the cells have ruptured and that the protein is denatured. The tube is then centrifuged. After centrifugation, the chelex resin and the denatured protein remain at the bottom of the tube and the DNA remains dissolved in an aqueous supernatant. The Chelex suspension is alkaline, has pH between 9.0 and 11.0, and as a result, the DNA that is isolated using this procedure is single stranded.
The major advantages of this method are: it is quick, takes approximately 1 hour; it is simple, multiple tubes are not involved and so mixing of samples is unlikely; cost is low; no harmful chemicals are used; and the DNA extract produced using this method is not of very good quality but it is sufficiently clean in most cases for downstream applications.
<H3>Silica-based DNA Extraction
This method is also called the 'salting out' procedure. It proceeds in several stages. The first stage of extraction involves breaking up of the cell membranes. In order to do this, the cells are incubated in a lysis buffer that contains a detergent along with the enzyme, proteinase K. The commonly used detergents are sodium dodecyl sulfate (SDS), Tween 20, Triton X-100 and Nonidet P-40. The lysis buffer breaks down the cell membranes and then breaks the cell. The protein structure can be interfered with at this stage by interfering with the hydrogen bonding. To do this, a chaotropic salt (Chaotropic agents are those which disrupt the structure and denature macromolecules). such as- M guanidine thiocyanate or 6- M sodium chloride is added either during or after cell lysis. This disrupts the protein structure by interfering with hydrogen bonding, Van der Waals interactions, and hydrophobic interactions. What is now left is a broken cell with disrupted protein structure. The chaotropic salt, in addition, also precipitates the cellular proteins (by dehydrating the protein) which are removed by centrifugation or ï¬ltration.
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The methods to isolate DNA after cellular disruption vary widely. DNA has high affinity for silica and glass particles in the presence of a chaotropic salt. This property of DNA is widely used for its extraction. The other cell components are removed from the solution. The DNA can then be released from the silica/glass particles by suspending them in water. Without the chaotropic salt, the DNA no longer binds to the silica/glass and is released into the solution.
The DNA yield is usually abundant and pure. Commonly used commercial kits, such as the Qiagen kits, exploit the salting-out procedure. However, the time taken is more and there is a chance of sample contamination.
<H3>Phenol-chloroform-based DNA extraction
The phenol-chloroform based DNA extraction method has been widely used in molecular biology. This method has been the mainstay of laboratories since its inception. However, it presents several problems. The phenol used is toxic and difficult to handle. The method also leaves a large number of impurities in the DNA which interfere with downstream PCR applications. Hence, the method has been slowly phased out since the mid 1990s. The advantage of the method is that it is cheap, however the drawbacks associated with the method do not permit its use.
Cell lysis is performed as described previously. Phenol-chloroform is added to the cell lysate and mixed. The phenol denatures the protein. The chloroform present in the mixture dissolves the lipids. The extract is then centrifuged. The precipitated protein forms a separate layer between the organic phenol-chloroform phase and the aqueous phase. The aqueous layer contains DNA in solution which is precipitated using ethanol. The DNA is then washed and can be used. .
If the procedure is done properly, clean DNA can be extracted. However, the method has several drawbacks including the toxic nature of phenol and the fact that the process is cumbersome and labour intensive.
<H2>DNA extraction from different tissues
The extraction of DNA from the spermatozoa is more complicated that the process of extraction of DNA from whole blood. The spermatozoa DNA is found in the head of the spermatozoa and the head to the spermatozoa is protected by the acrosome cap. This acrosome cap is rich in the amino acid cysteine. There are a large number of disulphide bridges between the cysteines in the acrosome cap. Proteinase K, which is a reagent used in the extraction process cannot break the disulphide bonds. This reduces the efï¬ciency of extraction. The addition of dithiothreitol (DTT), a reducing agent that breaks disulphide bonds, effectively increases the release of DNA from the spermatozoa.
The root of the hair shaft is rich in cellular material. DNA can be extracted from it using any of the commonly used techniques. The hair shaft, like the spermatozoa acrosome, is rich in disulphide bridges. Breakage of these disulphide bridges required mechanical grinding or as mentioned earlier, the addition of a reducing agent such as dithiothreitol. Once the disulphide bonds have broken, the proteinase K can digest the hair protein and release any trapped nucleic acids.
DNA extraction from soft tissues does not present a problem. The tissues need to be homogenised followed by lysis of the cells. This allows destruction of the protein structure and release of nucleic acids from the nucleus. The protocol for bones and other hard tissues is slightly different. Bones contain a large amount of positive ions and these ions have to be removed from the samples before extraction. If the ions are allowed to remain, they are likely to interfere with the downstream applications, mainly the PCR. Once the samples are partly processed, they are homogenized in lysis buffer using a mechanical homogenizer.
Commercial kits are also available for DNA extraction. The methods used will not be elaborated upon. A comparison between the kit and manual methods is elaborated in Table 3.1
Table3.1: Comparison of Kit based and Manual methods for DNA extraction
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<H2>Quantiï¬cation of DNA
After extracting DNA, an accurate measurement of its amount and an idea about its quality is desirable. Adding DNA to a PCR in correct amounts produces best quality results in the shortest possible time; adding too much or not enough DNA may result in a proï¬le that is difficult or even impossible to interpret.
<H3>Visualization on agarose gels
A relatively quick and easy method for assessing both the quantity and the quality of extracted DNA is to visualize it on an agarose gel. A 1% agarose gel is usually made. Quantiï¬cation standards can be run alongside the unknown samples to allow DNA concentrations to be estimated. If quantification standards are not available, the extracted DNA can be run on the gel without any standards. The gel shows the presence of the DNA which is visualized as a single bright band. Since the DNA has a high molecular weight, the DNA is seen very close to the well. If the DNA has degraded, multiple fragments are generated. These fragments are seen in the form of a smear.
The advantages of agarose gel electrophoresis are that it is quick and easy to carry out and also gives an indication of the size of the DNA molecules that have been extracted. The disadvantages are that quantiï¬cations are subjective, based on relative band intensities; it is difï¬cult to gauge the amounts of degraded DNA as there is no suitable reference standard; it cannot be used to quantify samples extracted using the Chelex method as this produces single stranded DNA and the ï¬‚uorescent dyes that intercalate with the double stranded DNA do not bind to the single stranded DNA.
DNA absorbs light maximally at 260 nm. This feature can be used to estimate the amount of DNA in an extract by measuring the absorption of light wavelengths between 220 nm and 300 nm. It is also possible to assess the amount of carbohydrates (maximum absorbance at 230 nm) and proteins (maximum absorbance at 280 nm) that may have been co-extracted with the DNA sample. The DNA is placed in a quartz cuvette and light is shone through; the absorbance is measured against a standard. The ratio of the absorbance of a clean DNA extract should be between 1.8 and 2.0 at 260 nm and 280 nm respectively. The major disadvantage is that it is difï¬cult to accurately quantify small amounts of DNA using spectrophotometry. The advantages as compared to the agarose gel quantification method is that the quantification of DNA is precise. It also gives an idea of the impurities which are present in the DNA sample.
Either ethidium bromide or DAPI (6-diamidino-2-phenylindole) can be used to visualize DNA in agarose gels. These chemicals ï¬‚uoresce at much higher levels when they intercalate with DNA. These chemicals can be used to stain the agarose gels. In addition, they can also be used as an alternative to UV spectrophotometry for DNA quantification. .
Extracted DNA is applied to a positively charged nylon membrane using a slot or dot blot process; a probe is then applied to the DNA, the probe being specific to human DNA. . A series of standards is applied to the membrane, and comparison of the signal from the extracted DNA with the standards allows quantiï¬cation. The advantage of hybridization-based methods is that the quantiï¬cation is speciï¬c to humans.
<H2>Nature of the Clinical Samples
Usually four types of biological material are available to the investigators for molecular studies:
Tissue removed from the patient, either as resection during surgery or as a small biopsy;
Cytology samples, including cells from fine needle aspiration biopsy or body fluids;
Blood, plasma, and serum products; and
Autopsy specimens. Autopsy specimens are the worst when it comes to tissue preservation. In general, the longer the postmortem time, the poorer the molecular quality of the tissue.
The pathologist receiving the tissue has three immediate options: he can either freeze it, or keep it fresh, or stabilize it in a fixative. Each of these generates a different type of specimen for analysis.
Tissue may be frozen directly at −80°C in a suitable medium, or by immersing in liquid nitrogen preferably in a medium such as isopentane. Frozen tissues procured for research may be homogenized for recovery of DNA, mRNA, and proteins. Processing of fresh tissue samples can proceed in two ways; either the cells are dissociated and the cells are used for the production of cell lines. Alternatively, the tissue sample maybe homogenized and DNA, RNA or protein maybe recovered from the tissue. Cytological specimens are smearedon a glass slide and either air dried or fixed in an alcohol based fixative.
As far as stabilisation of tissues using a fixative is concerned, it is important to remember that the prefixation time should be kept to a minimum. Although, this applies strictly to RNA and proteins, DNA is also preserved better if the tissue is immersed in a fixative as soon as possible. Formaldehyde is the commonest fixative used and therefore, its effect on DNA will be described here.
Studies of chemical reactions between formaldehyde and nucleic acids have shown that the reactions are similar to those observed in formalin-protein interactions. Formaldehyde initiates DNA denaturation and also causes several physical changes in the DNA. Thus, when compared to DNA isolated from frozen tissues, DNA isolated from formalin-fixed tissues shows a certain amount of sequence alterations. These sequence alterations are a result of cross linking of formalin with cytosine nucleotides on the DNA strands. As a result of this cross linking, the Taq polymerase is unable to recognize the cytosine and it then incorporates adenine in the place of guanine. This then results in the formation of an artificial C - T or G - A mutation. Up to 1 mutation artifact per 500 bases has been recorded. If the PCR product is sequenced following the PCR reaction, it may show the presence of a mutation. This mutation is a laboratory error and should be reflected as such. The overall rate of formalin-induced modification of DNA is dependent on the concentration, temperature, and pH of the fixative. Formaldehyde fixation at room temperature results in poor preservation of high-molecular weight DNA. The size of the extracted DNA is directly related to the fixation temperature, if the fixation temperature is higher, the loss of nucleic acids will be more. Up to 30% of nucleic acids may be lost during fixation. Tissues fixed in formalin at 4°C exhibit least amount of degradation of the nucleic acids.
An acidic environment can cause a decrease in the nucleic acid yield. Prolonged tissue hypoxia causes a decrease in the pH and this causes a decrease in the yield of nucleic acids. Similarly, if the tissues have been fixed in formalin at an acidic pH, some amount of formic acid is generated. This formic acid can also cause a decrease in the yield of nucleic acids. As compared to neutral buffered formalin, tissues fixed in formaldehyde solution at pH 3.0 had a greater number of artificial mutations.
In addition to the pH, presence of DNase in tissues is one of the factors that causes DNA degradation during fixation. Formaldehyde solution containing DNase-neutralizing ethylenediaminetetraacetic acid (EDTA) is a better fixative for preserving tissue DNA.
The speed of fixation depends on the rate of diffusion of fixative into the tissue and the rate of chemical reactions with various components. As a general rule, the longer the duration of fixation, the worse the quality of nucleic acids. Immediate microwave irradiation of tissues at ~60°C for 1 to 2 minutes has shown to preserve nucleic acids better. This is probably because of reduced enzymatic degradation and enhanced fixation. The average size of DNA extracted from tissues fixed in buffered formalin decreases with increasing fixation time.
In conclusion, criteria recommended in literature for the use of formaldehyde as a tissue nucleic acid fixative are as follows:
minimal prefixation time lag< 2 hours;
use of cold 10% neutral formalin;
low salt concentration;
cold fixation (at 4°C);
duration of fixation should be between 3 to 6 hours;
use of ethylenediaminetetraacetic acid (20 mmol/L to 50 mmol/L) as an additive;
Maintain pH and avoid a low pH environment.
In routine histopathology, the tissues obtained from a patients body are fixed in an appropriate fixative. The tissues are then processed and wax blocks are made. Sections are made from these wax blocks and the blocks are then stored in the archives. It is highly likely that a researcher might want to retrieve the wax blocks for nucleic acid analysis at a later date. The blocks provide investigators with valuable archival material which can be analysed using modern technology. However, there are issues of quality assurance and quality control. As mentioned earlier, there are several variables which are involved in nucleic acid preservation. It is not known whether storage of paraffin blocks and/or the histological sections under different conditions of temperature could prevent nucleic acid degradation.
<H1>RNA EXTRACTION: METHODS AND PRINCIPLES
In a cell, 80%--85% of the total RNA is contributed by ribosomal RNA (rRNA). However, the RNA which is a key player in the transcription process, messenger RNA (mRNA) contributes only 1%-5% of the total cellular RNA.
mRNA is heterogeneous both in size and sequence. It varies from few hundred bases to several kilobases in length. In most of the eukaryotes, mRNAs carry a long stretch of polyadenylate residues, that is, a poly (A) tail at their 3′ end. What we usually require in molecular medicine is mRNA.
Ribose residues of RNA have two hydroxyl groups, one each at 2′ and 3′ positions. The presence of two hydroxyl groups makes RNA much more chemically reactive than DNA. Therefore, RNA becomes prone to cleavage by contamination with RNases. Cells release RNases upon lysis. Moreover, RNases are even present on our skin. Therefore, the procedure of RNA extraction demands constant attention to prevent contamination of glassware and working bench area by RNases. Even the generation of RNases in the aerosols should be avoided. There are no simple methods available for RNAse inactivationand this compounds the difficulty in extracting RNA. Intra-chain disulfide bonds present in RNase, provide resistance against prolonged boiling and addition of denaturants. Moreover, addition of ion chelators (e.g. EDTA) is also ineffective, since RNases do not require divalent cations for their activity. In conclusion, it can be said that the best way to deal with RNases is to avoid them.
<H2>Principle of RNA extraction
RNA is usually extracted by the method developed by Chomczynski and Sacchi in 1987. This method is based on the use of three main components:
A chaotropic denaturing solution, such as guanidinium thiocyanate and guanidinium chloride.
When an aqueous sample is mixed with these three reagents and centrifuged, phase separation occurs such that there is an upper aqueous phase and a lower organic phase, with an interphase in between.
The aqueous phase contains almost all the RNA, which can be recovered as a precipitate by addition of isopropanol. The organic phase contains proteins, while, DNA is present in the interphase.
<H3>Treatment of reagents and glasswares with RNase inhibitor
Before attempting RNA isolation, it is important to treat the glassware and reagents with RNAse inhibitors. The most widely used RNase inhibitor is diethylpyrocarbonate (DEPC). To make all the solutions, glasswares and plastic wares RNase free, they are treated with DEPC. DEPC is a highly reactive alkylating agent and thus it abolishes the enzymatic activity of RNase.
Since RNases can even be released from our fingers, absolute precautions are required to avoid contamination with RNases. These precautions include wearing gloves and avoiding speech over open tubes. The working bench should be completely dust free. Aerosol-barrier tips should be used, so as to avoid any contamination of the reagents or samples by the RNase present in the form of aerosols. All the solutions should be prepared in the DEPC-treated water. All the organic liquids (phenol, chloroform and ethanol) should, essentially be of RNase free grade.
The first step in RNA extraction is cellular disruption. The most important factor for the purification of intact RNA is speed. At the very first step of the extraction process, the cellular RNase should be inactivated as fast as possible. Immediate destruction of the endogenous RNases significantly decreases the threat to the stability of RNA.
RNA molecules are very small, therefore, unlike DNA, they are less prone to damage by mechanical shearing forces. Various mechanical methods such as grinding, homogenization with a mechanical homogenizer, vortexing and sonication can be used to disrupt the tissue.
As soon as the tissue is crushed, the powdered tissue is transferred to a tube containing guanidinium thiocyanate solution, which is used as the extraction buffer. The extraction buffer should be at least five times the volume of the tissue. It must be added to the crushed sample as soon as possible to destroy the endogenous RNases.
Guanidinium thiocyanate is a strong chaotropic agent. It is a denaturant that disrupts cells, solubilize their components and denature endogenous RNases simultaneously. Since RNA is often tightly associated with proteins (e.g. rRNA with ribosomes), deproteinization is needed. Guanidinium thiocyanate demolishes the three-dimensional structure of the proteins, and converts them to a randomly coiled state. This allows RNA to come in its free form. A reducing agent like mercaptoethanol and detergents may also be used at this stage.
After adding the extraction buffer, the sample is mixed thoroughly by either vigorous shaking or vortexing.
To the tissue homogenate, phenol and chloroform-isoamyl alcohol are added and mixed properly. Phenol solubilizes the proteins, whereas chloroform dissolves lipids and leads to formation of a clear, upper aqueous phase that contains RNA; and a dark pink, lower organic phase, containing proteins and lipids. DNA remains at the interphase.
To the clear aqueous layer, iso-propanol is added to precipitate RNA. Precipitation at low temperatures (≤-20ËšC) is more effective. After precipitation, the RNA pellet is washed with 75% ethanol to remove the impurities. The pellet along with 75% ethanol can be either stored at -80ËšC for further use or dissolved in DEPC-treated water.
Now-a-days, guanidinium thiocyanate, phenol and chloroform are together commercially available in a solution form.
<H3>Dissolving the RNA pellet
Finally, the RNA pellet is dissolved in appropriate amount of DEPC-treated water. If the RNA pellet does not dissolve properly, the tube is kept in 55°C water bath for few minutes. This helps in dissolving RNA without affecting its quality. The RNA concentration can be estimated with the help of ultraviolet spectrophotometry.
<H3>Storage of RNA
The dissolved RNA is stored at -20°C (for short term) or -80°C (for long term storage).
The quality of mRNA in the tissue sample handled by the pathologist is the most important factor in obtaining the best possible diagnostic and prognostic information. If the starting point of the genomic tests is RNA, it is essential to ensure that the quality of RNA is good.
RNA is required in all cases of expression analysis. The best tissue from which RNA can be extracted is frozen tissue. The RNA extracted from frozen tissues can be used for applications like PCR of genome wide microarray expression analysis. In several laboratories, tissue is routinely frozen and preserved. This frozen tissue provides better nucleic acids than formalin fixed, paraffin embedded tissues. Frozen tissues allow a better preservation of nucleic acids which can even be used for Southern and Northern blotting where intact nucleic acids are essential.
In most oncology cases, however, pathology departments still rely on formalin-fixed, paraffin-embedded (FFPE) tissue as their standard method of preservation. These FFPE tissues can also be analyzed for expression of a limited number of select genes using the standard reverse transcriptase PCR or quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). FFPE tissue is especially useful when there are limited number of genes (around 100). However, it must be remembered that the mRNA in FFPE tissues is often highly degraded compared with the mRNA in frozen tissues. It is, therefore, not a good starting point for carrying out genomic tests.
<H1>EFFECT OF FORMALIN FIXATION ON mRNA QUALITY
Formaldehyde causes slight shrinkage and distortion of tissues. Even then, formaldehyde as 10% neutral-buffered formalin is still the most common fixative used by pathologists. Formalin is an excellent fixative for preserving tissue structure. It is also excellent for antigenic evaluation when one wants to look for specific antigens using immunohistochemistry. Unfortunately, the properties which make formalin an ideal fixative also cause it to degrade the mRNA in a random manner. There are several methods by which formalin destroys mRNA. Usually it causes destruction by adding monomethylol groups to the RNA bases. It also destroys RNA by cross linking the nucleic acids with proteins and by causing RNA fragmentation.
<H2>Danger of Delay: Enzymatic Degradation
mRNA is a short-lived molecule. mRNA should be a short lived molecule because its action is very transient. . Regulation of cytoplasmic RNA degradation is one of the cell's mechanisms for controlling gene expression. RNA degradation can be carried out by several methods including by altering the nutrient or hormone levels or by ischaemia and tissue hypoxia. As mentioned earlier, RNA is rapidly digested by the RNAses which are present in most tissues. mRNA begins to degrade within an hour of its removal from the body. That is why it is important to freeze the tissue immediately after it is removed from the body. If the tissue is not frozen immediately, it begins to degrade. Degradation occurs most commonly in tissues like the pancreas, gall bladder and the skin which contain high levels of endogenous RNAses.
Many eukaryotic mRNAs have half-lives of 30 minutes or less. There are certain mRNAs which are more prone to rapid degradation like mRNA which code for the cytokines and proto oncogenes. This is necessary because the cell has to maintain a regulatory mechanism. If cytokines are expressed in large amounts,the consequences can be catastrophic for the cell. For the clinician or the pathologist, it is of utmost importance to prevent RNA degradation and so rapid freezing is recommended. The extensive degradation of mRNA that occurs due to delay in fixation or preservation has been well documented.
<H2>Nature of Formalin Damage: Methylation, Cross-linking, Fragmentation
Formalin preserves tissue structure mainly by creating cross-links between proteins. Some intermolecular cross-linking of proteins with nucleic acids also occurs. Though this makes neutral-buffered formalin (10%) ideal for hardening tissues for later microscopic and IHC analysis, this also makes it difficult to extract intact mRNA from formalin-fixed tissues.
One way to break this cross linking is by using Proteinase K. The other method is by heating the tissue with guanidium which breaks the non covalent interactions which prevent RNA isolation and solubilisation. Both these methods are used to extract RNA from tissues. Unfortunately, neither of them can prevent the other major problem associated with fixation i.e nucleic acid fragmentation. RNA isolated from formalin fixed tissue is often fragmented with most of the fragments less than 300 bp in size.
As has been mentioned earlier, it is reiterated that freezing is far better than using formalin as a preservative. Immediate freezing in liquid nitrogen appears to be the best choice for preserving tissue for genomic analysis. In order to prevent tissue ischaemia and hypoxia which causes RNA loss, it is recommended that approximately 0.1 cm3 of the tissue should be snap frozen in liquid nitrogen within half and hour. 0.1 cm3 of tissue yields sufficient mRNA for most studies. After snap freezing, the tissue should be stored at at -80°C or below. If it is not possible to store the mRNA immediately, the tissue can be carried on ice and stored at -80°C. Repeated thawing of the tissue is not recommended but up to three freeze thaw cycles are permitted. Three freeze thaw cycles will not compromise theRNA integrity and it will not interfere with downstream applications. Other alternative is to store RNA in an RNA-friendly preservative. One of these solutions is called RNAlater. It precipitates RNases in an aqueous sulfate salt solution and thereby preserves intact RNA. Although RNA yields and specific gene RNA abundance with RNAlater are generally comparable with those seen with frozen tissue, freezing still may be preferred over RNAlater for RNA preservation.
For clinicians in institutions with access to proper equipment for freezing specimens, recognizing those special situations when such tissue acquisition and handling steps are prudent will be critical. In coming years, pathologists will need to work closely with their oncology colleagues to monitor a range of issues related to the impact of tissue handling on genomic expression profiling.