In fact, mRNA is the RNA of choice in reverse transcription and cDNA synthesis. There may be different amounts of mRNA in cells: from large quantities to just five copies per cell. RNA includes ribosomal RNA (rRNA) 80%, mitochondrial RNA (mtRNA), messenger RNA (polyadenylated – poly A + in eukaryotic cells) (mRNA) 1–5%, transfer RNA (tRNA), and microRNA molecules (miRNA). What accounts for the differences between the methods for extraction of DNA and RNA is their different stability. Nucleic acid degradation can result from hydrolysis of the 3′-5′ phosphodiester bonds catalyzed by metal complexes as well as from the spontaneous breakage of these bonds due to transesterification via a nucleophilic attack at the phosphorus atom by an adjacent 2′-hydroxyl group. It is important to provide appropriate conditions for nucleic acid extraction in order to avoid nucleic acid degradation due to oxidation by reactive oxygen species generated during respiration in vivo or, extracellularly, by mechanisms involving metal ions. The choice of method – in view of optimal time/quality balance – depends on the aim of the study, the type of analysis, the type of nucleic acid, and the cost. These methods yield initial nucleic acid samples of different purity and concentration depending on the original sample (bacteria, viruses, tissues). This is principally based on heat adsorption on silica membranes/beads, anion exchange chromatography, sedimentation/precipitation, and use of magnetic particles. The purpose of nucleic acid extraction methods is to disintegrate the cell envelope and achieve maximum elimination of lipids and proteins to obtain pure DNA and/or RNA.
Thus, nucleic acid extraction could be defined as a series of steps to obtain nucleic acid samples/materials of particular purity that are free of impurities and are suitable for different downstream application steps. Since nucleic acid extraction is a starting point in a vast array of downstream applications, the high quality of nucleic acids in the starting samples is a key factor for the success of the subsequent steps of analysis. Finally, the chapter outlines the application of nucleic acids in the diagnosis of various diseases, in scientific research, and bird sex determination by downstream applications such as restriction enzyme analysis, polymerase chain reactions (PCR, reverse transcription-PCR, real-time PCR), and different sequencing methods (Sanger, cycling sequencing, and next-generation sequencing). Other topics discussed here include comparative analysis of the use of these methods in DNA and RNA extraction from a variety of biological and clinical samples, as well as the relationship between the type of sample, the method used and the quality and amount of extracted DNA or RNA. This chapter describes the fundamental principles of different methods for nucleic acid sample preparation / nucleic acid extraction, such as column-based methods using silica membranes and traditional ones without a column purification procedure (commercially available or homemade). Nucleic acids, DNA and RNA, and their characteristics are discussed in other chapters of the book. Nucleic acids are not only a source of life but also a means of observing, understanding, and regulating it.