Experimental considerations

In this section, the experimental considerations for nucleic acids sample preparation is discussed. The following review aims to collect the information that helps in preparing reliable sample for intended applications and analysis.

To secure the quality of the analytical results, the first step is defining the parameters for sample preparation based on the complete workflow and the purpose. The following five issues should be clarified before starting the experiment.

1. Purpose of the intended application or analysis 2. The biomolecule that is of interest

3. Characteristics of the sample source used in the study 4. The detail of the analytical techniques used in the study

5. The capabilities of the analytical techniques and the criteria for optimal performance

• Sample collection

Starts from the sample collection stage, the quality of biomolecules start to decrease.

Therefore, the just prepared materials and samples are used in all our presented experiment. To those samples or materials that are not used immediately, they are stored in a -80°C refrigerator with the stabilising agent. When carrying out the mRNA isolation, the extreme cautiousness for minimising the introduction of exogenous RNases to the sample is needed. For the DNA isolation, the quality of the sample surely benefit the downstream analysis. Thus, all the DNA samples are processed in the well-cleaned biosafety cabinet (BSC).

To access the component inside the cell, the cultured cells are directly lysed and delivered to the following application or analysis. With some understanding of the basic characteristics of the cells of interest, the lysis protocol that efficiently disrupt the cell’s external barriers should preserve the integrity of the cell’s chemical components. This preservation is critical for molecules that will be studied in downstream applications.

Generally, the animal cells are easily disrupted due to the lack of the cell wall.

However, the variation between cell membrane compositions and the distribution of the molecules on it can make situation more complex. that is, an incomplete cell lysis or sample homogenisation leads to a reduced yield. In addition, it also increases the risk of problems in the following process. For example, the debris may clog the purification column or inhibit the enzymatic reactions like PCR. Besides, the nucleases and proteases may be liberated and activated while the cell disruption. The activated enzymes can extensively influence the eventual analysis of the target molecule.

Therefore, the sample should be protected from the action of such enzymes during the cell disruption and the subsequent purification. Particularly, it is crucial to inhibit the RNases and proteases to obtain the high-quality RNA and protein for the following

analysis. Table 1.1 summarises the most used methods and points out the corresponding appropriate sample type and target biomolecule. Generally, tender disruption are preferred when the cell of interest can be easily lysed (e.g. most of the cultured cells, blood cells, and some of the microorganisms). Tender disruption methods are also suitable when only one particular sub-cellular fraction is under consideration, like only cytoplasmic proteins or intact mitochondria are going to be analysed. Sometimes tender disruption methods are combined. For example, using the osmotic lysis then enzymatic treatment or freeze-thaw in the presence of detergent. When cells are less easily broken, the moderate disruption methods are employed. Usually, these methods involve the mechanical process to physically break the tissue.

Table 1.1 Methods for disrupting and homogenising cell for sample isolation.

protein isolation • simple, inexpensive

• inefficient for the cell

For most of the nano-medicine research, the cultured mammalian cells, which is easily disrupted, are widely used. In the case of cells grown in suspension, they are typically harvested by centrifugation, washed, and resuspended in the chosen lysis buffer. The buffer usually contains the relevant denaturants, inhibitors, and stabilisers. Overall, the use of vortex is enough to achieve the complete lysis. On there other hand, the adherent cells can be directly lysed in the culture plate with the same route mentioned above.

• Quality and purity considerations

The quality of the collected sample is important, since it directly represents the extent of the specific molecule in vivo. The poor quality shall lead to the misunderstanding of the cellular condition. In the RNA isolation, the degradation is the key concern. Thus, inhibition of the RNases is of leading importance in RNA isolation process. Together with the intact composition of the biomolecule, three-dimensional structure and the activity are also the important considerations. For example, the plasmid DNA with open circle or denaturation performs differently compared to the desired form. To ensure the quality of the isolated biomolecules, kinds of means can be used to determine the purity.

For example, the RNA integrity number (RIN) is a quality measurement for RNA. The output value 1 to 10 corresponding to the poorest to the highest quality (Schroeder et al., 2006). In the case of DNA, the quality can be measured by the Phred quality scoring (Ewing and Green, 1998, Ewing et al., 1998).

When preparing the sample, the requirement of the purity varies from the sample type, the target biomolecule, and the intended applications and analyses. In the case of the nucleic acids, the A₂₆₀/A₂₈₀ ratio is a basic but important index. Although the purity requirement differs from cases, thus there is no universal definition of purity. However, this ratio does provide the information about removal of the contaminants in the nucleic acid sample. This index is important because of many biological analyses are sensitive to the contaminants. In order to obtain the reliable analytical results, interfering compounds like salts, polysaccharides, and non-target molecules have to be removed.

Table 1.2 includes a list of common contaminants and means to deal with.

Table 1.2 Common contaminants in the sample preparation.

• General guidelines for cell disruption and sample homogenisation

Specifically, the following description of preparation is recommended by the previous literature. Firstly, a gentle process is usually a benefit for preparation. It is because the cell may denature, the target biomolecule may be sheared, and the protease and nuclease may be release under the vigorous procedure. Secondly, any extraction process should be performed as quickly as possible. The condition at sub-ambient temperature and the pre-chill equipment for keeping samples in preferable. Besides, the presence of the appropriate buffer to maintain the pH and ionic strength shall stabilise the sample.

Thirdly, using the additives to inhibit the nucleases and proteases is important for the experiment, especially the one that related to the gene expression monitoring. Fourthly, considering the possibility of the cross-contamination and take the precautions. For instance, the quantitative PCR is susceptible to the contamination from the residue of previous DNA amplifications. To avoid that, the aerosol pipette tips are should be used.

In addition, the samples are usually prepared at the different place from the one doing PCR analysis. Fifthly, the DNA can be stored at the room temperature for several hours, at 4℃ for several days(with the aids of EDTA to inhibit the activity of DNase is necessary). For long-term storage, the DNA should be frozen at -20℃ or -80℃. In the case of RNA, since it is very sensitive to the RNases, which is often found in the environment. Creation of an RNase-free working environment is very important. The wear gloves at all times during the preparation is necessary. Changing them frequently is also recommended. Lastly, the isolated RNA should be hold on the ice during the use or frozen in -80℃ for long-term storage.