Extraction and quality control of nucleic acids in qPCR experiments

Due to the simple operation, fast detection speed, high sensitivity and good specificity of qPCR experiments, qPCR has become the gold standard for nucleic acid quantitative experiments. At the same time, it is also widely used in other fields, such as qualitative and quantitative detection of pathogens, viral microorganisms, gene copy number detection, gene expression analysis, gene fusion analysis, SNP analysis, identification of exogenous genes in genetically modified foods, risk assessment of cancer recurrence, and applications in forensics, making it widely used in life sciences, agronomy, molecular diagnosis and medicine.

In previous issues, we shared with you the principles and development of qPCR, the design process of primers, 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 links in conducting qPCR experiments, and are also one of the important links 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 according to MIQE, here are some guidance and suggestions for you:

1. Sample Collection, Processing, and Preparation

Sample collection may be the first source of experimental variation. Whether the amount, location and time of sample collection are consistent is an important consideration for the correctness of the results. Therefore, when collecting samples, we must emphasize the consistency of amount, location and time. In addition, the collection environment and storage method are also very critical links, especially for RNA experiments. Compared with DNA, RNA is easily degraded, so it is easy to be degraded by improper sample collection and processing methods, thus affecting the accuracy of the results. For RNA samples, it is recommended that samples should be collected in a relatively clean environment and operated at low temperature or on ice. After sample collection, liquid nitrogen should be used for quick freezing and stored at -80℃; if the Trizol method is used to extract RNA, the sample can be placed in Trizol, homogenized and stored at -80℃; in addition, the sample can be stored in an RNA protective agent (there are many ready-made kits on the market now) to avoid RNA degradation. For DNA samples, the processing method and storage conditions are relatively simple.

2. Nucleic Acid Extraction

Nucleic acid extraction is the second key step. The extraction efficiency, amount and purity of nucleic acid will affect the subsequent experimental results. Therefore, choosing the correct extraction method and quality control method is very important.

(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 method, namely phenol/chloroform extraction method. It mainly uses the principle that DNA is soluble in water but insoluble in organic solvents, and that proteins can be denatured and precipitated in the presence of organic solvents. After nucleic acids and proteins are separated according to their different reactivity to phenol and chloroform denaturation, DNA is collected by ethanol precipitation under high salt conditions. This method can be used to extract tissue samples from Jiaotong University and obtain relatively high yield and quality. However, this method is time-consuming and labor-intensive, and requires operators to have certain experience. It cannot be extracted in large quantities and is difficult to automate. In addition, organic solvents are potentially harmful to the health of operators.

b. Membrane column method This method mainly utilizes the principle of solid-phase binding of DNA molecules to adsorb DNA onto the adsorption membrane (such as glass cellulose membrane) of the centrifugal column, and centrifuges to remove molecules such as proteins and RNA. This method can extract a variety of sample types and obtain high-quality DNA (both genomic and small fragment DNA) for subsequent analysis. Since the membrane column method is easy to operate, it is suitable for large-scale and high-throughput processing. However, when the starting material is over-homogenized (such as the tissue sample of Jiaotong University) or incompletely homogenized, it may cause clogging of the adsorption membrane, resulting in reduced yield or potential contamination.

c. Magnetic bead adsorption extraction method Biomagnetic beads are superparamagnetic microspheres with extremely small particle sizes. They have rich surface active groups and can be coupled with various biochemical substances and separated under the action of an external magnetic field. According to the different groups coated on the magnetic beads, magnetic beads can be divided into epoxy magnetic beads, amino magnetic beads, carboxyl magnetic beads, aldehyde magnetic beads, thiol magnetic beads and silicon-based magnetic beads. Among them, epoxy magnetic beads, amino magnetic beads and carboxyl magnetic beads can be used for the separation of various proteins or antibodies, and thiol magnetic beads can be used for the separation of heavy metal substances. The magnetic beads used for DNA separation and extraction are silicon-based magnetic beads. The extraction principle is to use the superparamagnetism of silicon oxide nano-microspheres. Under the action of guanidine hydrochloride, guanidine isothiocyanate and an external magnetic field, DNA molecules can be specifically and efficiently adsorbed. Compared with the membrane column extraction method, this method eliminates the influence of sample blocking the membrane, and is simple to operate and easy to automate. In addition, the free magnetic beads have a larger binding amount with nucleic acids, and the specific binding makes the nucleic acid purity higher.

(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 mainly utilizes the characteristics of Trizol reagent containing phenol, guanidine isothiocyanate and other substances, which can quickly destroy cells and inhibit nucleases released by cells. Under the action of isopropanol, RNA molecules in the sample can be completely precipitated. This method is the most classic and traditional, and is suitable for most sample types, especially tissue samples that are difficult to lyse. However, it should be noted that this method may also introduce inhibitors that affect subsequent PCR enzymatic reactions during the operation (such as hemoglobin in the blood, humic acid and fulvic acid in plant samples, and EDTA, heparin, chlorophenol, chloroform, etc. introduced during the experiment). These will directly affect the subsequent reverse transcription and qPCR, resulting in deviations in the results.

b. Membrane column method This method uses a series of lysis solutions to lyse tissues or cells and inhibit RNase at the same time. The silica gel membrane specifically adsorbs RNA and then rinses it multiple times to remove DNA, protein and other impurities. Finally, RNA is eluted with a low-salt solution. Compared with the Trizol extraction method, the membrane column method is simpler and easier to automate, and is suitable for large-scale and high-throughput processing.

c. Magnetic bead adsorption extraction Depending on the type of magnetic beads used, this method can extract total RNA and mRNA of the sample respectively. The principle of extracting total RNA from the sample by magnetic bead method is basically the same as that of extracting DNA from the sample by magnetic bead method. Both use the affinity adsorption ability of silicon-based magnetic beads for nucleic acids to separate nucleic acids under the action of a high salt environment and an external magnetic field. However, unlike DNA extraction, before using magnetic beads to extract RNA, a special lysis solution is required to pre-treat the sample to remove RNase and separate the RNA layer for subsequent total RNA extraction. Different from silicon-based magnetic bead extraction, the magnetic bead method uses magnetic beads coated with avidin to extract sample mRNA. After the sample to be extracted is annealed and combined with the biotin-labeled oligo (dT) probe, it interacts with the magnetic beads coated with avidin to achieve the purpose of separating mRNA. Compared with the membrane column method, the use of magnetic bead method to extract RNA eliminates the influence of sample blocking the adsorption membrane, and the operation is simpler. However, due to the high preparation requirements and cost of RNA adsorption magnetic beads, there are not many commercial kits on the market.

3. Nucleic acid quality control

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

It is important to quantify the concentration of DNA/RNA in the extracted sample. For absolute quantification, the sample must be within the range of the standard curve. As shown in Figure 1, the standard curve is linear, but this trend cannot be extended indefinitely. Once the template concentration exceeds the limit of the PCR reaction, the amplification efficiency will decrease and the quantitative linearity will decrease. When the template concentration is too low, background contaminants may be mistaken for amplification signals, and the signal-to-noise ratio is insufficient. At the same time, it is also risky to extrapolate the standard curve to a larger range, so it is best to confirm that the sample is within the appropriate concentration range before performing qPCR.

For relative quantification, such as gene expression by RT-qPCR, lower amounts of template will increase error. Methods such as SNP genotyping will also generate more reliable qualitative data when the appropriate amount of template is used for analysis. As shown in Figure 2, the signal intensity of the unknown sample should be similar to that of the standard sample. Too little template will make allele expression quantities unreliable or even unusable.

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

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

Commonly used nucleic acid quantitative quality control methods

At present, the most commonly used quantitative methods in laboratories are spectrophotometry (quickdrop (MD), NanoDrop (Thermo)), microfluidics analysis (Agilent Technologies' Bioanalyzer, Bio-Rad Laboratories' Experion), capillary gel electrophoresis (Qiagen's QIAxcel), or fluorescent dye detection. The principle of spectrophotometry is that nucleic acids, nucleotides and their derivatives all have conjugated double bond systems and can absorb ultraviolet light. The ultraviolet absorption peaks of RNA and DNA are at a wavelength of 260nm. Generally, at a wavelength of 260nm, the light absorption value of a solution containing 1μg RNA per 1ml is 0.022~0.024, and the light absorption value of a solution containing 1μg DNA per 1ml is about 0.020. Therefore, the light absorption value of an unknown concentration of RNA or DNA solution at 260nm can be measured to calculate the nucleic acid content therein. This method is simple and rapid. If the sample is mixed with a large amount of substances that can absorb ultraviolet light, such as nucleotides or proteins, the photometric error will be large, so it should be removed in advance. NanoDrop and other UV spectrophotometers use UV absorbance for detection, which cannot distinguish between DNA, RNA, degraded nucleic acids, free nucleotides and other impurities.

Microfluidic analysis (2100) uses microfluidic technology to separate samples. By adding voltage, samples bound to fluorescent dyes are separated in microscopic etched channels on the chip. The separation is based on the different mobility of nucleic acid molecules, and the dye is made to fluoresce by excitation light so that it can be detected by the instrument. The functional relationship between DNA migration time and size is calculated based on the known molecular weight and content ladder, and the molecular weight and concentration of the sample are calculated according to the formula.

Fluorescent dye detection, which uses fluorescent dyes to detect the concentration of specific target molecules. It uses specially developed fluorescent detection technology that uses Molecular Probes® dyes that only fluoresce when bound to DNA, RNA or protein. These fluorescent dyes can only emit fluorescent signals when bound to specific target molecules, even in the presence of free nucleotides or degraded nucleic acids. Because the dye method only detects the concentration of the target molecule (not contaminants), this specificity can obtain very accurate results.

These methods can give different results, so it is unwise to compare results using different methods. The recommended method for quantifying DNA/RNA is the fluorescent dye method, which is the best method for detecting low concentration samples and is also the most accurate method among the quantitative methods. It is recommended to use the same method for all samples in any case.

b. Purity test

Contaminants remaining during the extraction process will greatly affect downstream analysis, and many contaminants can be detected using quickdrop and NanoDrop spectrophotometers. Figure 3 shows a purified DNA sample (A), as well as the same sample contaminated by guanidine (B) and phenol (C). Through preliminary testing, both contaminated samples showed specific spectral characteristics of nucleic acids. In fact, the 260/280 ratio of the phenol contaminated sample is mostly normal.

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

Although a rough spectrum cannot diagnose a problem, it may help identify if a problem exists and narrow down the source of the problem. Many contaminants absorb at wavelengths around 230 nm or less; some can cause problems in downstream applications. In addition to checking the 260/280 ratio and the rough 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 valley in the spectrum – it should be around 230 nm. Contaminants that absorb at shorter wavelengths will generally shift the valley 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 have a characteristic spectrum, such as phenol. However, many contaminants have a similar characteristic spectrum: absorbance at 230 nm or less. 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 small 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 using a spectrophotometer to detect purity, electrophoresis can also be used for detection. The electrophoresis diagram can be used to check whether there is RNA, DNA, and protein contamination. If there is genomic DNA contamination in the RNA sample, it can be treated with DNase I. In addition, designing primers across introns will also reduce the impact of genomic contamination (I wonder if you still remember the content of our second issue); if the sample contains protein and other impurities, it is recommended to use KAPA or Beckman magnetic beads for nucleic acid purification.

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

a. Integrity test

Integrity testing is an essential step, especially for RNA samples, as RNA degradation will greatly affect our experimental results. For integrity testing, we can use gel electrophoresis, or 2100, LabChip for testing, and the latter is the gold standard for integrity testing.

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

What should we pay attention to in experiments with degraded nucleic acid samples? Especially for rare and precious tumor FFPE samples or forensic samples, the quality of nucleic acids in these samples is relatively poor and the degree of degradation is relatively serious. For qPCR experiments of these samples, we can reduce the fragments of amplified products to reduce the impact of the sample itself on the experiment.

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