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1. A diagnostic kit in vitro for assessing a histopathological stage of tumour and presence of metastases, wherein the assessing is performed by detecting in a biological sample expression of a sequence comprising SEQ ID NO: 1 through PCR Real-time and detecting one or more genes express in cancer stem cells chosen among CD133, c-myc, Nanog, Oct4, TCFL1, ZIC1, KLF5, HMGA1, HMGB3, wherein the kit comprises:
a) a pair of amplification primers for detecting the sequence comprising SEQ ID NO:1, including:primer Forward:(SEQ ID NO: 3)CGGGAATTCCCAGAGGACACCTCCAC; andprimer Reverse:(SEQ ID NO: 4)CGGCTCGAGCCGAGGGTCTAACCCAG b) a pair of control primers for normalizing an expression value of the sequence comprising SEQ ID NO: 1; c) one or more of a pair of primers together with a probe for detecting the one or more genes, including:cd133:primer Forward:(SEQ ID NO: 49)TCCACAGAAATTTACCTACATTGGAA;primer Reverse:(SEQ ID NO: 50)GGGTCAGCAGAGAGCAGATGA; andprobe:(SEQ ID NO: 51)AGTATGATTCATACTGGTGGCTGGGTGGCcmyc:primer forward:(SEQ ID NO: 52)GCTGGATTTTTTTCGGGTAGTG;primer reverse:(SEQ ID NO: 53)TAGTTCCTGTTGGTGAAGCTAACG; andprobe(SEQ ID NO: 54)CAGCAGCCTCCCGCGACGnanog:primer forward:(SEQ ID NO: 55)CCGAAGAATAGCAATGGTGTGA;primer reverse:(SEQ ID NO: 56)GCATCCCTGGTGGTAGGAAGA; andprobe:(SEQ ID NO: 57)CAGCACCTACCTACCCCAGCCTTTAoct 4:primer forward:(SEQ ID NO: 58)AACCCGGAGGAGGTAGTCCTT;primer reverse:(SEQ ID NO: 59)AATAACCTACCCACAAATGTCATTCA; andprobe:(SEQ ID NO: 60)CATGCATGAGTCAGTGAACAGGGAAtcfl1:primer forward:(SEQ ID NO: 61)CCAAGGAAGAGAACGTTGACATAG;primer reverse:(SEQ ID NO: 62)CCGAGGGAGCAGGATCAAG; andprobe:(SEQ ID NO: 63)AGGCTCTTTGTGTTTTTCCTTGTCTTTTGTCCzic 1:primer forward:(SEQ ID NO: 64)CGCGCTCCGAGAATTTAAAG;primer reverse:(SEQ ID NO: 65)CGGTCACAGCCCTCAAACTC; andprobe:(SEQ ID NO: 66)TCCACAAAAGGACGCACACAGGGKLF5:Primer forward:(SEQ ID NO: 67)TTTAACCCCACCTCCATCCTATG;Primer reverse:(SEQ ID NO: 68)GGCAGGGTGGTGGGTAAATT; andprobe:(SEQ ID NO: 69)TGCTTCTAAACTGGCAATTCACAATCHMGA1:Primer forward:(SEQ ID NO: 70)CCCCAGGCAGACCTTATATGAG;Primer reverse:(SEQ ID NO: 71)TGCTTGAGGTCAGGAGTTCGA; andprobe:(SEQ ID NO: 72)CATGGGAGTCCCACCGTATTGTCCA; andandHMGB3:primer forward:(SEQ ID NO: 73)CGCTATGATCGGGAAATGAAG;primer reverse:(SEQ ID NO: 74)TGGCCTTTTGGGAGCATTAG; andprobe:(SEQ ID NO: 75)ATTATGGACCAGCTAAGGGAGGCAAG;and d) a pair of primers and a probe for normalizing an expression value of the genes in c), including:primer Forward:(SEQ ID NO: 81)GCCAACCGCGAGAAGATG;primer Reverse:(SEQ ID NO: 82)ACAGCCTGGATAGCAACGTACA; andprobe:(SEQ ID NO: 83)CCCAATCATGTTTGAGACCTTCAAC. |
