Nucleic acid extraction and quality control in qPCR experiments

Due to its simple operation, rapid detection, high sensitivity, and excellent specificity, qPCR has become the gold standard for nucleic acid quantification. It is also widely used in other fields, such as qualitative and quantitative detection of pathogens and viruses, gene copy number detection, gene expression analysis, gene fusion analysis, SNP analysis, identification of exogenous genes in genetically modified foods, cancer recurrence risk assessment, and forensic applications, making it widely used in life sciences, agronomy, molecular diagnostics, and medicine.

In previous issues, we shared with you the principles and development of qPCR, the primer design process, and the design of experimental methods. This issue will share with you the extraction and quality control of nucleic acids. Sample collection, processing, and preparation, nucleic acid extraction, and quality control are the initial steps in conducting qPCR experiments and are also one of the important steps that determine the accuracy of qPCR experimental results. Correct sample collection and processing methods, as well as effective nucleic acid extraction and strict quality control are the guarantees for accurate qPCR experimental results. So, based on MIQE, here are some guidelines and suggestions for you:

1. Sample Collection, Processing, and Preparation

Sample collection may be the primary source of experimental variability. Consistency in sample quantity, location, and timing is crucial for accurate results. Therefore, consistent sample collection is crucial. Furthermore, the collection environment and storage method are crucial, especially for RNA experiments. Compared to DNA, RNA is more susceptible to degradation, making improper sample collection and handling more susceptible to RNA degradation, thus affecting the accuracy of results. For RNA samples, it is recommended that samples be collected in a clean environment at low temperatures or on ice. After collection, samples should be snap-frozen in liquid nitrogen and stored at -80°C. For RNA extraction using the Trizol method, samples can be placed in Trizol, homogenized, and stored at -80°C. Alternatively, samples can be stored in an RNA protectant (many pre-made kits are available) to prevent RNA degradation. Handling and storage conditions for DNA samples are relatively simple.

2. Nucleic Acid Extraction

Nucleic acid extraction is the second key step. The extraction efficiency, quantity, and purity of nucleic acids will affect subsequent experimental results. Therefore, choosing the right extraction method and quality control method is crucial.

(1) DNA extraction method: The most commonly used methods for DNA extraction are organic solvent extraction method, membrane column method and magnetic bead adsorption extraction method.

a. Organic solvent extraction, also known as phenol/chloroform extraction. This method primarily utilizes the principle that DNA is readily soluble in water but insoluble in organic solvents, and that proteins can be denatured and precipitated in the presence of organic solvents. Nucleic acids and proteins are separated based on their different reactivity to phenol and chloroform denaturation, and DNA is then collected by ethanol precipitation under high-salt conditions. This method can be used to extract tissue samples from Jiaotong University, achieving relatively high yield and quality. However, this method is time-consuming and labor-intensive, requiring operator experience, making large-scale extraction impossible and difficult to automate. Furthermore, organic solvents pose potential health risks to operators.

b. Membrane column method: This method primarily utilizes the principle of solid-phase binding of DNA molecules, adsorbing DNA onto an adsorption membrane (such as glass cellulose membrane) in a centrifuge column. Proteins, RNA, and other molecules are then removed by centrifugation. This method can be used to extract a variety of sample types, yielding high-quality DNA (both genomic and small DNA fragments) for subsequent analysis. Due to its ease of operation, the membrane column method is suitable for large-scale and high-throughput processing. However, when the starting material is over-mixed (such as the tissue samples from Jiaotong University) or incompletely homogenized, the adsorption membrane can clog, resulting in reduced yield or potential contamination.

c. Magnetic Bead Adsorption Extraction Method: Biomagnetic beads are ultra-paramagnetic microspheres with extremely small particle sizes. They possess a rich array of surface-active groups that can couple with various biochemical substances and achieve separation under the influence of an external magnetic field. Depending on the groups coated on the beads, they can be categorized as epoxy-, amino-, carboxyl-, aldehyde-, thiol-, and silica-based. Epoxy-, amino-, and carboxyl-based magnetic beads can be used to separate various proteins or antibodies, while thiol-based magnetic beads can be used to separate heavy metals. Silica-based magnetic beads are used for DNA isolation and extraction. The extraction principle utilizes the superparamagnetic properties of silica nanoparticles. Under the influence of guanidine hydrochloride, guanidine isothiocyanate, and an external magnetic field, DNA molecules are specifically and efficiently adsorbed. Compared to membrane column extraction, this method eliminates the effect of sample clogging the membrane and is simple to operate and easy to automate. Furthermore, the free magnetic beads bind to a greater amount of nucleic acids, and this specific binding results in higher nucleic acid purity.

(2) RNA extraction method: Currently, the RNA extraction methods include Trizol extraction method, membrane column method, and magnetic bead adsorption extraction method.

a. Trizol extraction method: This method primarily utilizes the properties of the Trizol reagent, which contains substances such as phenol and guanidine isothiocyanate, which rapidly disrupt cells and inhibit nucleases released by cells. The isopropanol-based method allows for complete precipitation of RNA molecules in the sample. This is the most classic and traditional method and is suitable for most sample types, especially difficult-to-lyse tissue samples. However, it should be noted that this method may introduce inhibitors that affect the subsequent PCR enzymatic reaction (such as hemoglobin in blood, humic acid and fulvic acid in plant samples, and EDTA, heparin, chlorophenol, and chloroform introduced during the experiment). These inhibitors can directly affect subsequent reverse transcription and qPCR, leading to distorted results.

b. Membrane column method: This method uses a series of lysis buffers to lyse tissues or cells while simultaneously inhibiting RNases. The RNA is then specifically adsorbed onto a silica gel membrane, followed by multiple rinses to remove DNA, proteins, and other impurities. Finally, the RNA is eluted using a low-salt solution. Compared to the Trizol extraction method, the membrane column method is simpler and easier to automate, making it suitable for large-scale and high-throughput processing.

c. Magnetic bead extraction: Depending on the type of magnetic beads used, this method can be used to extract both total RNA and mRNA from samples. The principle behind magnetic bead extraction of total RNA is essentially the same as that used for DNA extraction: both utilize the affinity of silica-based magnetic beads for nucleic acids, separating them in a high-salt environment and under the influence of an external magnetic field. However, unlike DNA extraction, RNA extraction using magnetic beads requires pre-treatment with a special lysis buffer to remove RNases and separate the RNA layer for subsequent total RNA extraction. Furthermore, unlike silica-based magnetic bead extraction, mRNA extraction using magnetic bead extraction utilizes avidin-coated magnetic beads. The sample to be extracted is annealed with a biotinylated oligo(dT) probe, which then interacts with the avidin-coated magnetic beads to isolate mRNA. Compared to membrane column extraction, magnetic bead extraction eliminates the risk of sample clogging the adsorption membrane and is more convenient. However, due to the high preparation requirements and cost of RNA-adsorbing magnetic beads, commercially available kits are limited.

3. Nucleic acid quality control

a. Why do we need to do quantitative quality control?

Quantitative analysis of DNA/RNA concentrations in extracted samples is crucial. For absolute quantification, samples must fall within the range of the standard curve. As shown in Figure 1, the standard curve is linear, but this trend is not indefinite. Once the template concentration exceeds the limits of the PCR reaction, amplification efficiency decreases, and the linearity of quantification diminishes. If the template concentration is too low, background contaminants may be mistaken for amplification signal, resulting in an insufficient signal-to-noise ratio. Furthermore, extrapolating the standard curve to a wider range is risky. Therefore, it is best to confirm that the sample is within the appropriate concentration range before performing qPCR.

For relative quantification, such as gene expression analysis by RT-qPCR, low template amounts can increase error. Methods such as SNP genotyping can also generate more reliable qualitative data when analyzed with appropriate template amounts. As shown in Figure 2, the signal intensity of unknown samples should be similar to that of the standard sample. Excessive amounts of template will render allele expression unreliable or even unusable.

Figure 2: Examples of SNP genotyping using appropriate template amounts (left) and low template amounts (right). When using appropriate template amounts, the allele signals for the unknown sample are very clear, while with low template amounts, the allele signals are unreliable or unusable.

Additionally, it is advisable to use equal amounts of DNA/RNA when comparing different samples.

Commonly used nucleic acid quantification quality control methods

Currently, the most commonly used quantitative methods in laboratories include spectrophotometry (QuickDrop (MD), NanoDrop (Thermo)), microfluidics analysis (Agilent Technologies' Bioanalyzer, Bio-Rad Laboratories' Experion), capillary gel electrophoresis (Qiagen's QIAxcel), or fluorescent dye detection. Spectrophotometry is based on the fact that nucleic acids, nucleotides, and their derivatives all possess conjugated double bonds that absorb ultraviolet light. The UV absorption peaks for RNA and DNA are at 260 nm. Generally, at 260 nm, the absorbance of a solution containing 1 μg of RNA per ml is 0.022–0.024 nm, and the absorbance of a solution containing 1 μg of DNA per ml is approximately 0.020 nm. Therefore, measuring the absorbance at 260 nm of an unknown RNA or DNA solution can be used to calculate the nucleic acid content. This method is simple and rapid. However, if the sample contains significant amounts of UV-absorbing substances, such as nucleotides or proteins, photometric errors can be significant, so these substances should be removed beforehand. NanoDrop and other UV spectrophotometers use UV absorbance for detection, which cannot distinguish between DNA, RNA, degraded nucleic acids, free nucleotides, and other impurities.

Microfluidics (2100) uses microfluidics to separate samples. By applying voltage, samples bound to fluorescent dyes are separated in microscopically etched channels on the chip. Separation is achieved based on the size and mobility of nucleic acid molecules. Excitation light causes the dyes to fluoresce, enabling detection by the instrument. The relationship between DNA migration time and size is then calculated using a ladder with known molecular weight and content, and the molecular weight and concentration of the sample are calculated using a formula.

Fluorescent dye detection uses fluorescent dyes to measure the concentration of specific target molecules. It utilizes specially developed fluorescence detection technology, employing Molecular Probes® dyes that fluoresce only when bound to DNA, RNA, or protein. These fluorescent dyes emit a fluorescent signal only when bound to their specific target molecule, even in the presence of free nucleotides or degraded nucleic acids. Because dye-based detection methods only measure the concentration of the target molecule (and not contaminants), this specificity allows for highly accurate results.

These methods can produce different results, so comparing results using different methods is unwise. Fluorescent dye-based methods are recommended for quantifying DNA/RNA. These methods are best for detecting low-concentration samples and are the most accurate of the various quantification methods. It is recommended that all samples be tested using the same method in all cases.

b. Purity testing

Contaminants left over from the extraction process can significantly impact downstream analysis, and many contaminants can be detected using QuickDrop and NanoDrop spectrophotometers. Figure 3 shows a purified DNA sample (A) and the same sample contaminated with guanidine (B) and phenol (C). Preliminary testing revealed that both contaminated samples exhibited spectral signatures specific to nucleic acids, and the 260/280 ratio of the phenol-contaminated sample was generally normal.

Figure 3: Spectra of uncontaminated purified DNA (A), and the same DNA sample contaminated with guanidine (B) and phenol (C). Note that the troughs and peaks in the contaminated samples shift, typically appearing at 230 nm and 260 nm, respectively.

While a rough spectrum cannot diagnose a problem, it may help identify the presence of a problem and narrow down its source. Many contaminants absorb at wavelengths around 230 nm or shorter; some can cause problems in downstream applications. In addition to examining the 260/280 ratio and the general shape of the spectrum, we recommend the following:

• Check the 260/230 ratio – A low ratio indicates the presence of a contaminant in the sample that absorbs at 230 nm or less.

• Check the wavelength of the dip in the spectrum – it should be around 230 nm. Contaminants that absorb at shorter wavelengths will generally shift the dip to the right.

• Check the wavelength of the peak in the spectrum – DNA and RNA should be at 260nm. Contaminants that absorb at longer wavelengths will generally shift the peak to the right.

Some contaminants, such as phenol, have characteristic spectra. However, many contaminants share a common characteristic: absorbance at 230 nm or shorter. Absorbance at 230 nm may indicate a problem with the sample or the extraction process, so it is important to consider both. For example, a high A230 value (a low A260/A230 ratio) may be caused by:

• polysaccharide residues (usually found in plants);

• Phenol residues from nucleic acid extraction;

• Residual guanidine (commonly used in column-based kits);

• Glycogen residues for nucleic acid precipitation.

In addition to testing purity using a spectrophotometer, electrophoresis can also be used to detect RNA, DNA, and protein contamination. If the RNA sample is contaminated with genomic DNA, treatment with DNase I can be performed. Designing primers that span introns can also reduce the impact of genomic contamination (remember our discussion in Part 2). If the sample is contaminated with other impurities such as protein, KAPA or Beckman magnetic beads are recommended for nucleic acid purification.

Figure 4: Nucleic acid gel electrophoresis to detect RNA or DNA contamination.

a. Integrity test

Integrity testing is essential, especially for RNA samples, as RNA degradation can significantly impact experimental results. We can perform integrity testing using gel electrophoresis, 2100, or LabChip, the latter being the gold standard for integrity testing.

Figure 5. RNA integrity testing using the 2100 or LabChip.

What precautions should be taken when conducting experiments with degraded nucleic acid samples? This is especially true for rare and valuable FFPE tumor samples or forensic samples, where the nucleic acid quality is poor and the degree of degradation is severe. For qPCR experiments with these samples, we can reduce the fragmentation of the amplified product to minimize the impact of the sample itself on the experiment.

Here are the advantages and disadvantages of different quality inspection methods: