Thirdly, allele-specific (AS) primers and a FAM-containing TaqMan probe (Bio-Rad, USA) were used for JAK2V617F detection, whereas primers for the control gene were mixed with a HEX-containing probe to quantify JAK2 exon 21 ( Figure 1C), and the calculation of mutant AB was determined by the ΔCt method. They were used to establish quantification standards. The plasmid pair had the same size (3306 bps) and could be easily manipulated with identical copy numbers. In the second step, the exon 21 control sequence, along with either one of the target regions, were cloned into a yT&A cloning vector (Yeastern Biotech, Taiwan) to generate two standard plasmids ( JAK2_WT_Ctrl_yT&A and JAK2_V617F_Ctrl_yT&A, representing 0% and 100% mutant AB, respectively) ( Figure 1B). It improved the efficacy of copy number normalization by directly calibrating equal copies of target DNA with the reference control within the same gene. Firstly, we decided to select JAK2 exon 21 as our reference control ( Figure 1A). To improve the accuracy of JAK2V617F detection and quantification, we made some refinements to the allele-specific real-time polymerase chain reaction (qPCR) assay and created a novel method. 1 Although DNA from JAK2-mutated UKE-1 and HEL cells are commonly used for this purpose, 2, 3 none of these cells are considered ideal, as the HEL cells carry multiple copies of JAK2, and UKE-1 cells do undergo clonal evolution with increasing JAK2 copies during in vitro cultures. 1 The study also concluded by delineating the importance of using well-defined, accurate standards to refine JAK2 quantitative assays. Unfortunately, there were significant discrepancies in the allele burden (AB) quantification when blinded samples were tested in a multicenter study. Since its discovery, many diagnostic techniques have been applied in its detection. You can find out how to avoid them below.JAK2V617F mutation is one of the key driver mutations in myeloproliferative neoplasms (MPN). These cookies and other technologies capture data like your IP address, when you viewed the page or email, what device you were using and where you were. We use various advertising partners, including Amazon, Facebook, and Google. These cookies are used to track your activity on the BenQ website and other websites across the Internet, help measure the effectiveness of our advertising campaign and deliver advertisements that are more relevant to you and your interests. See list of performance and advertising cookies To opt-out of Hotjar collecting data, you can disable tracking completely by following link:. To opt-out of SessionCam collecting data, you can disable tracking completely by following link:. To opt out of certain ads provided by Google you can use any of the methods set forth here or using the Google Analytics opt out browser add-on here. You can control the information provided to Google, SessionCam and Hotjar. If you want to opt-out of advertising cookies, you have to turn-off performance cookies. We also use Google Analytics, SessionCam and Hotjar to track activity and performance on the BenQ website. ![]() These cookies help to improve the performance of BenQ. Performance cookies and advertising cookies
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