circRNA-qPCR引物设计的方法

circRNAqPCR引物设计的方法1、通过cirbase数据库()获得circRNA的序列，如hsa_circ_0014629：进入如下页面点击fasta,获取序列,如下:选择splicedhsa_circ_0014629|NM_001037533|GON4LCCTCCTCAGTTTGTGGATATCCACCTTGAAGAAGATGATTCCTCAGATGAAGAATACCAGCCGGATGATGAAGAAGAAGATGAAACTGCTGAAGAGAGCTTATTGGAAAGTGATGTTGAAAGCACTGCTTCATCTCCACGTGGGGCAAAGAAATCCAGATTGAGGCAGTCTTCTGAGATGACTGAAACAGATGAGGAGAGTGGCATATTATCAGAGGCTGAGAAAGTCACCACACCAGCCATCAGGCACATCAGTGCTGAGGTAGTGCCCATGGGGCCCCCGCCCCCTCCAAAGCCGAAACAGACCAGAGATAGTACTTTCATGGAGAAGTTACATGCGGTAGATGAGGAGCTGGCTTCCAGTCCAGTCTGCATGGATTCTTTCCAGCCCATGGATGACAGTCTCATTGCATTTCGAACGCGTTCTAAGATGCCCCTGAAAGATGTTCCCCTGGGCCAATTAGAGGCAGAGCTCCAAGCTTTGATGGGAGCTATGCAGCTGATTGAAGACTTCAGCACACATGTCAGCATTGACTGCAGCCCTCATAAAACTGTCAAGAAGACTGCGAATGAATTTCCCTGTTTGCCAAAGCAAGTGGCTTGGATTCTGGCCACAAGCAAGGTTTTCATGTATCCAGAGTTACTTCCAGTGTGTTCCCTGAAGGCAAAGAATCCCCAGGATAAGATCGTCTTCACCAAGGCTGAGGACAA2、取首尾各150bp进行拼接，末尾150bp作为拼接后的首序列，如下：GACTGCGAATGAATTTCCCTGTTTGCCAAAGCAAGTGGCTTGGATTCTGGCCACAAGCAAGGTTTTCATGTATCCAGAGTTACTTCCAGTGTGTTCCCTGAAGGCAAAGAATCCCCAGGATAAGATCGTCTTCACCAAGGCTGAGGACAACCTCCTCAGTTTGTGGATATCCACCTTGAAGAAGATGATTCCTCAGATGAAGAATACCAGCCGGATGATGAAGAAGAAGATGAAACTGCTGAAGAGAGCTTATTGGAAAGTGATGTTGAAAGCACTGCTTCATCTCCACGTGGGGCAAAG3、将该部分300bp用primer-blast进行引物设计，具体参数如下：=BlastHome4、选择引物对。注：PCR产物包含back-splicing位点Sequence(5'-3')TemplatestrandLengthStartStopTmForwardprimerATTCTGGCCACAAGCAAGGTPlus20446360.18ReverseprimerCACAAACTGAGGAGGTTGTCCTMinus2216514460.16Productlength122