Extraction

The first steps of any extraction process are tissue isolation, disruption, and cell lysis. Again, the specific protocol required depends on the sample. When a large portion of fresh or fixed solid tissue is available, it is important to select appropriate areas for subsequent harvest of nucleic acid. For collection of genomic DNA or RNA, tissue cannot be autolyzed or necrotic. Focal necrosis is common in many solid tumors, and sampling of these areas provides little or no intact nucleic acid. The his-tological complexity of a solid tissue sample should also be considered. Many tissues, lesions, and tumors are composed of multiple cell types and morphological areas. It is possible, and can be critical, to isolate nucleic acid from a single one of these areas. Careful dissection, and techniques like selective ultraviolet radiation fractionation (20) allow even small cell groups to be isolated and processed.

3.1. PARAFFIN AND BLOOD Paraffin-embedded tissues require deparaffinisation prior to nucleic acid extraction. A variety of methods exist that employ heat or solvents like xylene to remove paraffin (21). Heat-based protocols are simple and require only a microwave or thermal cycler. More precise temperature control might be possible with a thermal cycler, but either system should prove successful. Some direct comparisons suggest that yields from solvent-based techniques are lower than those using heat (22).

Virtually all nucleic acid obtained from blood samples, barring hematologic pathology, is leukocyte derived. Isolating white blood cells by centrifugation, therefore, could optimize purification yield and reduce reagent requirements. A simple method for this employs Ficoll lymphocyte separation medium (21). Other protocols allow successful nucleic acid preparation directly from whole blood (23). These techniques could prove quicker and involve fewer steps.

3.2. LYSIS/MEMBRANE DISRUPTION To purify nucleic acid from tissue samples, it is first necessary to disrupt cellular and nuclear membranes. This is efficiently accomplished with a detergent, often sodium dodecyl sulfate (SDS). The large amount of protein present in cell or nuclear lysates can make DNA or RNA purification difficult, so most methods employ proteolytic agents during this step. Proteinase K is frequently used for this purpose (6,14,21,24).

3.3. ORGANIC EXTRACTION Traditional nucleic acid purification from lysate is accomplished by phenol-chloroform extraction. Variations of this basic protocol rely on the separation of protein into the organic phase and nucleic acid into an aqueous phase. It is important that the phenol pH lie within a range of 7.8-8.0 to prevent nucleic acid from remaining in the organic phase. Even at this pH, RNA with a long poly(A) tail or tract might partition with phenol. The addition of isoamyl alcohol to the mixture prevents this, and reduces RNase activity (final ratio of phenol: chloroform: isoamyl alcohol of 25:24:1).

A key step in the organic extraction is emulsification of organic and aqueous phases. When low-molecular-weight DNA is desired, this emulsion can be achieved by vortexing. Higher-weight nucleic acid (>10 kb), however, is vulnerable to shearing forces and might tolerate only gentle shaking or rotation. The use of large-bore pipets will also reduce shearing during transfer of material, and limiting the number of transfers will also facilitate high-molecular-weight nucleic acid recovery. Extremely high-weight DNA required for pulsed-field gel electrophoresis might require cell lysis and DNA purification within an agarose plug. Digestion and removal of cellular proteins is accomplished over the course of days, leaving large and intact DNA within the agarose. This method does not employ phenol or chloroform as protein solvents.

The requirements for most molecular assays are met by conventional organic extraction, followed by ethanol precipitation. Adequate purity and yields could require serial phenol-chloroform extractions of the aqueous phase. The presence of visible protein at the interface of organic and aqueous materials warrants another round of extraction. Additionally, yield can be optimized by vigorously mixing Tris-EDTA (TE) buffer with the discarded organic phase ("back extraction"). Extra nucleic acid can be taken from the TE after subsequent centrifugation.

It is critical that the final aqueous nucleic acid solution be free of phenol and protein contamination. Care when removing the aqueous phase and repeated cycles of chloroform extraction prior to ethanol precipitation will prevent phenol contamination. In the presence of ethanol and monovalent cations, DNA or RNA precipitates out of solution at temperatures near 0°C. A variety of salts can be used as a cation source. Perhaps the most common is sodium acetate, and this is suitable for most organic extraction protocols. Other salts have unique advantages and disadvantages. Ammonium acetate reduces dNTP coprecipita-tion but can inhibit subsequent assays requiring nucleic acid phosphorylation. Sodium chloride is useful when samples are contaminated with SDS (24). As discussed earlier, lithium chloride facilitates RNA precipitation, a technique useful in removing heparin contamination.

3.4. INORGANIC EXTRACTION As an alternative to organic purification, inorganic techniques reduce exposure to hazardous reagents while producing purified nucleic acid of comparable quality. Many commercial kits employ inorganic purification methods, including salt precipitation, adsorption to silica surfaces, and anion-exchange chromatography protocols. Many of these principles are also easily applied without commercial kits. Removal of contaminating protein by precipitation and centrifugation prior to ethanol DNA precipitation, for example, gives good yields with excellent purity (25).

Especially popular are purification systems based on the binding of nucleic acid to silica or glass particles in the presence of chaotropic agents. The chaotropic agent GITC is useful for inhibiting troublesome nuclease, but it also promotes nucleic acid binding to silica or glass media (26,27). Nucleic acid elu-tion after washing can be accomplished with a low-salt aqueous buffer. Commercial systems based on this technique produce high-quality, high-purity nucleic acid preparations with improved safety and speed. There are many variations on this theme, depending on the manufacturer, and many laboratories choose to avoid the trouble of organic purification by investing in these standardized and modestly priced kits.

3.5. RNA EXTRACTION In general, variations of these techniques can be applied to both DNA and RNA purification. RNA isolation, however, demands extra care. Most forms of RNA are labile, and RNase is a frustratingly frequent contaminant of laboratory reagents and equipment. A few simple techniques and principles will help prevent degradation problems. First, reagents and equipment used for RNA preparation should be dedicated to that purpose. This becomes especially important if the laboratory also does DNA purification. RNase is frequently used in DNA work, and contamination of reagents and reusable equipment will affect subsequent RNA preparations.

Perhaps the greatest risk of RNase contamination comes from the laboratory worker's skin. It is critical that clean gloves by worn at all times and changed frequently if contact with potentially contaminated equipment is necessary.

Fresh out of the package, most sterile pipet tips and other disposable materials can be considered RNase free. Other tools might require treatment to destroy contaminating RNase. Glassware, reagent spatulas, and other equipment can be pre-treated by incubation at 37°C in a solution of 0.1% diethylpy-rocarbonate (DEPC). DEPC is a strong RNase inhibitor. In addition to inhibiting RNase, however, DEPC can also car-boxymethylate nucleic acid purine residues. After incubation, therefore, the materials must be autoclaved to remove any remaining DEPC before use in RNA preparation. As an alternative, glassware can be baked at 150°C for 4 h or plastic materials can be soaked in 0.5 M NaOH for 10 min. Use of NaOH requires subsequent rinsing and autoclave treatment. Any of these methods will reduce RNase activity on reusable equipment (21).

Decontamination of reagent solutions can also be accomplished by adding DEPC to a concentration of 0.1%. Caution should be exercised, however, with solutions containing amines that will react with DEPC. Tris buffer is a common example. Tris from a freshly opened or dedicated container can be added after autoclaving DEPC-treated water or solutions. To make Tris-EDTA for RNA storage, for example, a solution of EDTA can be DEPC treated and autoclaved before adding RNase-free (uncontaminated) Tris. Subsequent pH adjustments must also be made with RNase-free reagents.

3.6. DNA MICROARRAY The recent development of DNA microarray technology is also worth mentioning for its particular demands on nucleic acid extraction. In brief, cDNA transcribed from a pool of cellular mRNA is hybridized to an array of thousands of distinct DNA sequences fixed to a glass slide. Because the cDNA is transcribed with fluorescent or radioactive tags ,the relative quantity of hybridized cDNA, and therefore original mRNA, can be assessed. Generally, the gene expression profile of one cell source is simultaneously compared to a reference cell source (transcribed with a second fluorescent tag) as an internal control. This is a powerful new tool, with a rapidly growing body of literature describing its techniques and applications. One of the primary technical limits of microarray technology is the quality and quantity of RNA

available from the test and reference cells (28). Contamination of nucleic acid preparations with protein, lipid, or carbohydrate can interfere with reverse transcriptase or mediate nonspecific array hybridization. A variety of mRNA purification procedures are available, and a review of public domain protocols suggests that most laboratories employ commercial kits to purify total RNA and then isolate the mRNA fraction. Total RNA can be obtained efficiently with both silica-based techniques (RNeasy; Qiagen, Valencia, CA) or precipitation procedures (TRIzol; Life Technologies, Rockville, MD). Subsequent mRNA purification is best accomplished via binding to oligo dT fixed on a solid medium or column. A number of representative protocols are available on the World Wide Web (www.microarray.org, www.nhgri.nih.gov/DIR/Microarray, and cmgm.stanford.edu/pbrown).

As genetic testing is increasingly used for clinical work, other technologies for rapid, large-scale genetic analysis will become important. The Invader® System (Third Wave Technologies, Madison, WI), for example, is an automated system that can be used to test for clinically important genetic sequences such as the Prothrombin G20210A and Factor V Leiden mutations. This system employs a combination of proprietary enzymes and specific oligonucleotide probes to generate a fluorescent signal that is amplified in a linear manner from the target DNA. No PCR amplification is required, and as little as 100 ng of target genomic DNA (or total RNA) is sufficient for testing. This method is, however, sensitive to phenol, chloroform, and highsalt concentrations, so a careful inorganic purification method is generally recommended. As more testing and detection technologies become available, the demands on purified DNA or RNA could change and will likely become less stringent.

100 Pregnancy Tips

100 Pregnancy Tips

Prior to planning pregnancy, you should learn more about the things involved in getting pregnant. It involves carrying a baby inside you for nine months, caring for a child for a number of years, and many more. Consider these things, so that you can properly assess if you are ready for pregnancy. Get all these very important tips about pregnancy that you need to know.

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