| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | The recombinant plasmids containing target sequences were obtained from Genscript Biotechnology Co., Ltd (Nanjing, China). RNA Clean Kit was purchased from TIANGEN Biotech Co., Ltd. (Beijing, China). SmaI, ProtoScript II Reverse Transcriptase and Bst 2.0 WarmStart DNA polymerase were purchased from New England Biolabs Inc. (USA). T7 in vitro transcription kit was obtained from Biomics Biotechnologies (Nantong, China). HPLC purified DNA primers (Table S1, ESI†) and the sequencing data were obtained from Shanghai Sangon Biotech (Shanghai, China). Recombinant RNase inhibitor, Recombinant DNase I, dNTPs, TaKaRa Taq™ Hot Start Version and Ribonuclease H (RNase H) were obtained from Takara Biomedical Technology Co., Ltd. (Beijing, China). Betaine was purchased from Sigma-Aldrich (Shanghai, China). SYBR Green I (20× stock solution in DMSO, 20 μg mL−1) was gained from Zhishan Biotechnology Co., Ltd. (Xiamen, China). A549, 293T, HL-60 and HeLa cells were purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China). | Get A Quote |
Alternative splicing is a ubiquitous and crucial process in cellular processes and has a specific linkage with diseases. To date, developing cost-effective methods with high sensitivity and specificity for detection of splicing variants has been needed. Herein, we report a novel splicing variant assay based on specifically designed reverse-transcription loop-mediated isothermal amplification. After reverse transcribing the splicing variant into cDNA, four DNA primers are specifically designed to recognize six distinct regions. The four DNA primers can hybridize with corresponding sequences for extension and strand displacement DNA synthesis to form stem-loop DNA and then LAMP amplification is started. The propo... More
