About PCR internal reference gene selection strategy

2022-10-01 10:05:44

_{^{The choice of internal reference genes Many junior experimenters rarely think about this problem, because there are only two choices in front of them, Î²-actin and GAPDH, and even in many people's long-term perceptions, the understanding of internal parameters only stays. In the case of a relatively high and stable housekeeping gene, it is an important reference for the relative expression of the molecular level. As for why you choose these two, there are no other internal parameters. People often answer â€œSorry,â€ I don't care." Or "My predecessors used it like this. Other documents are written like this. Is there a problem?".}}

^{Is there a problem? BioTNT wants to scream here "Yes, and it's a big problem."} ^{When people detect a decrease in the expression of a gene or improve the results, you don't know whether its expression has changed, or because The amount of expression caused by other reasons such as the size of the sample or the loss of RNA caused by the operation, so it is necessary to simultaneously detect another gene as a reference at this time. This additional gene is the internal reference gene.}

^{Therefore, from our own scientific requirements, the ideal internal reference will have several conditions, 1, the expression is large; 2, stable expression in different types of cells and tissues; 3, expression levels and cell cycle and cells Whether activation is irrelevant; 4, is not affected by any endogenous or exogenous factors; 5, there is no pseudogene to avoid contamination amplification of genomic DNA. Please note that this is the ideal internal reference. Let's take a look at our common classic internal reference. Is it really ideal?}

^{The first is beta-actin, beta actin. Let's first take a look at what actin is. The so-called actin actin is an important cytoskeletal protein of cells. It can be roughly divided into six types, four of which are different in muscle tissue, including Î±-skeletal (skeleton). ), cardiac (heart), smooth (smooth) muscle actin and Î³-smooth muscle actin, the other two widely distributed in various tissues, including Î²-actin and Î³-non-muscle actin. The distribution of these different subtypes is different, and there is little distribution of beta-actin in muscle tissue. The myocardium is mainly Î±-cardiac muscle actin. Obviously, if my research model is heart or bone, Î²-actin is obviously not suitable as an internal parameter. Studies have shown that when cells are transformed into malignant, the expression level of Î²-actin mRNA is increased. It is obvious that this is easy to proliferate with tumor cells, and the characteristics of migration are theoretically correct. BioTNT is usually contacted with tumor tissue. In the experiment of the sample, although the wet weight of the tissue has been controlled, the difference in the expression of Î²-actin between the normal group and the tumor group is still quite large, which further limits the application range of Î²-actin, and Î²-actin is also a number. Not many people should pay attention to the internal reference of pseudo-gene pollution. With the deepening of the research on Î²-actin, it is often considered to be stable in expression, and the Î²-actin with high expression is actually not so stable, including Î²-actin in some visceral tissues of liver tissue. Good choice.}

^{The second is GAPDH. Let us look at GAPDH, glyceraldehyde-3-phosphate dehydrogenase, a reaction enzyme in the glycolysis process, which is also highly expressed in almost all tissue cells, and the expression is almost constant, which is indispensable in the cellular metabolic pathway. a gene, but also because it plays a non-negligible role in glycolysis, leading to its obvious influence on all endogenous and exogenous factors involved in the metabolic process, for example, the most In the typical cell division stage, the expression of GAPDH is significantly changed in different cell cycles. For the same reason, the rapidly dividing and proliferating tumor cell samples are also difficult to use with GAPDH as a constant expression internal reference, and also in related foreign sources. The mRNA expression levels of GAPDH were also stimulated by factors such as hypoxia, insulin, dexamethasone, mitogen, epidermal growth factor, etc., which made GAPDH not as stable as Î²-actin in some experiments.}

^{Now, the problem is very clear. The two internal references that we used to rely on in the past have more or less deadly defects in some places. For example, the study of tumor cells seems to be unable to use any conventional internal reference. How to solve it? Therefore, BioTNT will cite the commonly used internal parameters in 20 here, and a small one of them will be exemplified.}

^{Third, 18S rRNA and 28S rRNA. Since rRNA is synthesized by an RNA polymerase different from mRNA synthesis. Therefore, the regulation of rRNA synthesis is independent of mRNA. Under various conditions affecting mRNA expression, the levels of various r RNAs rarely change. In tumor samples, it may be a good choice to use rRNA as an internal reference. However, rRNA also has many problems. For example, when the target gene in purified mRNA is quantified, rRNA cannot be used for standardization because it is removed during purification. For example, rRNA does not include poly(A) structurally. The tail, so theoretically it cannot be reverse transcribed in cDNA synthesis with oligo(dT) as a primer. But here, BioTNT has tried to reverse-transcribe rRNA with oligo(dT) as a primer, and can successfully amplify cDNA, and the expression is not low, so BioTNT has been confused about this, but as far as rRNA itself is concerned The internal reference to use this point, BioTNT is still a small recommendation, in the special case of less sample size, sample quality is slightly lower, etc., using rRNA as an internal reference is better than Î²-actin and GAPDH.}

^{Â Â Â Â Â}

^{Fourth, other internal parameters. Some researchers have studied 67 cases of liver tissue and found that UBC (ubiquitin) and HMBS (bilirubinogen deaminase) are the best choice for liver tissue expression analysis. Comparison of Î²-actin, Î²2-MG, and PBGD (bilirubinogen deaminase) in leukemia, normal, and malignant tumors revealed that Î²2-MG is most suitable as an internal reference. PRKG1 and TBP are suitable for controlling the quality and yield of RNA in B and T cells and their tumors. Therefore, different internal reference genes have the potential to become suitable internal organs under different conditions. In a given set of specimens or experimental conditions, the determination of the expression levels of two or more internal controls in parallel helps to detect and reduce systematic errors.}

^{In summary, when we further conduct internal reference screening and research on different kinds of housekeeping genes, we will find that the internal reference world is more complicated than we think. When we study the object from pronucleus to eukaryotic, from From cell to tissue, from normal to worse, all the changing conditions are a screening for our internal reference. There is no universal internal reference suitable for all conditions and sample types. BioTNT thinks that it is double internal reference or even three internal reference or four internal reference. Experimenting is theoretically more scientific and less error-prone.}

^{For further research on internal reference, BioTNT has not been developed here. In fact, BioTNT has seen data obtained by selecting 4 internal parameters as the average value for qPCR in foreign countries for a more reliable correction. The value of this approach proves that some people have realized and tried this more scientific approach. BioTNT is only a word, but only hope that more people can realize the importance of internal reference before the experiment. Early circumvention may occur because the experimental data is not meaningful due to improper selection of internal parameters.}

^{In addition, if you are interested, you can find some reviews or papers about the internal reference.}

Striped Bonito

Striped Bonito,Sarda orientalis,Fresh Striped Bonito,Fresh Sarda orientalis

Zhoushan Boda Aquatic Products Co.,Ltd , https://www.baida-aquatic.com