The blood circulation system in the human body is like the holy river of India – the Ganges River. The nutrients carried in the river nourishe the ancient land; the washing and taking away the filth on the ground, people's life is completed in the river. Such a Ganges River can be imagined, the river is full of various substances, there are wastes and nutrients, as long as you want to be able to fish from the river. The same circulatory system is like the Ganges River; the flowing blood is like the Ganges water, the body's required cuts are transported by the blood, and the waste generated by the body is also transported by the blood. It is also conceivable that the blood components are all-encompassing. If we want to know how pure the earth is or how serious the pollution is, just take a scoop of water to check it to know the answer; and the same want to understand the human physiological condition, take a drop of blood to test it can also know the clue, but do these tests It is not untargeted. It is necessary to know which substances are so-called pointers. These pointers in the living body are known biomarkers. The research and application of biomarkers has been carried out in a variety of ways. Screening for various metabolic diseases has been widely used, and there have been some good biomarkers in cancer screening, such as AFP (Alpha-fetoprotein). Along with the discovery and research boom of microRNAs (miRNAs), about 1000 miRNAs have been discovered and expressed in humans. The physiological functions of miRNAs regulating gene expression in humans are also well known, and the high stability of miRNAs relative to protein markers. Sex, along with the rapid detection technology, makes the miRNAs in the blood become the target of a new generation of biomarkers. The content of this paper is mainly based on the experience accumulated by the laboratory in the operation of serum miRNA extraction and chip experiments, and share with the reader some of the problems encountered in the experiment and the solutions.
The composition of miRNA in the blood Where does the miRNA in the blood come from? Where are you going? What are the physiological functions of these miRNAs? These three questions are the concerns of many researchers. In fact, these problems are not very With clear answers, we know that miRNAs are endogenous short-fragment RNAs with a length of about 17 to 23 nucleotides. miRNAs can bind information RNA (mRNA) to induce degradation of information RNA, thereby regulating the expression of information RNA and relative proteins. . According to the disclosure of some papers 1 and the actual operating experience of the laboratory, the content of miRNA in the blood of normal people is extremely small, but a large amount of miRNA can be extracted in blood samples prepared by patients and samples. Based on the reasonable speculation of these observations, the source of miRNA in the patient's serum is likely to be an intracellular miRNA that is excreted due to tissue necrosis or apoptotic rupture caused by disease, and a chip map of the gene expression through patients and normal people. In comparison, the results also tend to have differences in performance or not, rather than the increase in specific miRNA expression; in other words, we observed that the number of patients' miRNA expression increased in related studies, and the analysis was in normal people and patients. The expression of miRNA, the high and low performance of the performance is the second representation. Therefore, the logic and methods of data analysis must also be adjusted, but these ectopic expressions of miRNAs do not affect their potential to be a marker for diagnosing disease and prognosis. Some studies have indicated that these miRNAs, which are not often expressed in the circulatory system, can be a good marker for disease diagnosis. For example, miR-141 can be used as an indicator of prostate cancer, and miR-499 and miR-208 are associated with myocardial infarction. miR-122 is associated with liver damage caused by drugs.
The challenge of miRNA extraction in serum
1. Preparation of the sample: Is the serum (serum) or plasma (plasma) good?
Clinically, the preservation and preparation of blood samples are selected according to the subject of the test. Both serum and plasma are components from the blood sample, but the ingredients are essentially different depending on the preparation method. According to the preparation method, the serum is a supernatant obtained by blood sedimentation through a blood coagulation reaction. Plasma is the liquid after the blood cells are removed by centrifugation without blood coagulation. However, it has been pointed out in the literature that the total amount of miRNA extracted from the same person's serum and plasma samples is significantly higher than the plasma extraction, which means that the preparation method is also Will affect the extraction of miRNAs, it is speculated that these miRNAs may be due to coagulation reactions, substances released after platelet or red blood cell rupture, including miRNA, although most of the real-time quantitative polymer chain reaction (q-PCR) verification results show that plasma There is no significant difference in the performance profile of miRNAs with blood. However, this existing risk may need to be considered in the study design. Therefore, in theory, plasma samples are more suitable as biomarkers.
2. Is the preparation or storage of the sample available?
It is known that the source of miRNA in serum or plasma may come from intracellular and extracellular, especially blood cells (red blood cells or white blood cells, etc.), so improper treatment will cause contamination of the sample, change the composition and expression of miRNA, resulting in research results. The deviation, especially the hemolysis of red blood cells, has a great impact, not only causing erythrocyte miRNA contamination, but also large fragments of mRNA in red blood cells can cause quantitative misalignment and false positives due to degradation. Therefore, it is also necessary to avoid the action of blood cell rupture when preparing the sample, such as blood drawing with too fine needle, or hemolysis due to improper storage of the sample, or the patient's own septic or hemolysis caused by disease or treatment may also be listed. Considerations for inclusion in the assessment. In addition, the use of anticoagulants must avoid the use of heparin-containing coagulation tubes. Heparin is known to inhibit reverse transcription and polymerase (polymerase) reactions. If the specimen is to be verified by q-PCR, it must be avoided. . The same blood sample also contains components that inhibit reverse transcription and polymerase, and if it is not removed cleanly, it will cause subsequent experiments to fail.
miRNA extraction method Basically, the miRNA in the blood is also RNA, but the fragment is short, so the extraction method and principle are similar to those of other RNA extraction. Currently, there are many extraction kits available on the market, and the extraction method is Organic solvents such as: Trizol LS, or through the column (column) can have a good recovery rate, the extraction method can refer to the April 2008 Hualian News – RNA extraction. Only one point is to remind the reader that the use of organic solvent extraction (phenol-chloroform) is prone to salt and organic solvent residues, which will affect the quantification and inhibition of subsequent experimental operations, so the operation must avoid the extraction of organic solvents, can increase Wash the alcohol residue one or two times to remove residual salts. Of course, the use of column extraction can easily avoid the risk of these potential problems.
Quantification of miRNAs In the case of successful extraction, serum and plasma contain only a small amount of RNA, and the total amount of 1 mL of blood may be in the ng or even pg level. This result means that the current brightness meter is used. It is not feasible to measure OD (for example, NaroDrop: the concentration to be tested needs to be as high as 10 ng/uL), and the Agilent bioanalyzer 2100 can only perform qualitative tests and cannot do accurate quantification. To examine the current quantitative methods used, there are probably: (1) adding an external control nucleic acid (spike) to an equal volume of serum or blood sample, and after the extraction step, the total amount of extraction can be corrected by detecting the added spike and Efficiency, as a difference in calibration efficiency due to extraction efficiency or error. However, this only applies to the perfect preparation of the sample and the control extraction technology. For the sample with different individual performance and the quality of the sample preparation, there is no way to achieve the effect of control; (2) OD is measured by the brightness measurement Quantitative, but unless the amount of extraction reaches the level of confidence of the brightness meter, these values ​​are probably false signals, not enough to accept the letter; (3) using the amount of internal control gene expression as a correction. However, according to the research, the number of miRNAs in serum or plasma is very different. The number of miRNAs in normal subjects is 123, and the highest is 296, which can be up to twice. The gap, together with the absence of suitable internal control genes, can be used as a control group. The commonly used internal control genes, such as U6 or SNORD RNAs, are highly variable and do not apply. Therefore, the relative quantification of miRNA by the results of the Agilent Bioanalyzer 2100; or the extraction of miRNAs from equal volumes of serum and the equal extraction of the experiments seems to be a more credible approach, but it depends on the design of the experiment.
Conclusion
When we understand the design of the experiment and the expected results, avoiding the possible interference factors, after completing the chip experiment and getting the results, you may have to review the original data before deciding which method to analyze the data, for example; Assuming that the control group only exhibits 200 miRNAs, but the patient group exhibits 400, the often used chip calibration methods such as median scaling or 75th percentile may not be applicable. Which kind of analysis is ideal? There may be no standard answer, and it must be based on experimental design and review data. This is a long story and another story. If interested readers can consult our biological information. Analyze the professional team. The above is a laboratory experiment on miRNAs in serum in the laboratory. The accumulated little attention is very helpful and I hope to be helpful to readers who are working on or preparing for such research.
Reference material
(1) Wang K., et al., Comparing the MicroRNA Spectrum between Serum and Plasma. PLoS One. 2012
(2) _Huang Z., et al., Plasma microRNAs are promising novel biomarkers for early detection of colorectal cancer. Int J Cancer. 2010 Jul 1;127(1):118-26.
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