Method of diagnosing, monitoring, staging, imaging and treating gastrointestinal cancer
a gastrointestinal cancer and imaging technology, applied in the field of gastrointestinal cancer detection, diagnosis, staging, imaging and treating cancer, can solve the problems of recurrence and metastasis, bowel obstruction and bowel perforation are indicators of poor prognosis, and much rarer types of cancer
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example 1
Relative Quantitation of Gene Expression
[0136] Real-Time quantitative PCR with fluorescent Taqman probes is a quantitation detection system utilizing the 5′-3′ nuclease activity of Taq DNA polymerase. The method uses an internal fluorescent oligonucleotide probe (Taqman) labeled with a 5′ reporter dye and a downstream, 3′ quencher dye. During PCR, the 5′-3′ nuclease activity of Taq DNA polymerase releases the reporter, whose fluorescence can then be detected by the laser detector of the Model 7700 Sequence Detection System (PE Applied Biosystems, Foster City, Calif., USA).
[0137] Amplification of an endogenous control is used to standardize the amount of sample RNA added to the reaction and normalize for Reverse Transcriptase (RT) efficiency. Either cyclophilin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ATPsy6 (ATP synthase 6), or 18S ribosomal RNA (rRNA) is used as this endogenous control. To calculate relative quantitation between all the samples studied, the target RNA l...
example 2
Expression of Cln114 in E. coli
[0154] Cln114 was amplified by polymerase chain reaction (PCR) using colon cDNA libraries and Cln specific primers. The amplified DNA fragment encoding amino acid number 1 (Met1) to amino acid number 323 (Ile323) of Cln114 was subcloned in a T7 RNA polymerase-based system (pET-21d) for expression in E. coli. In addition to Cln114 DNA coding sequence (Met1-Ile323), codons for six histidines, flanking the COOH-terminus of Cln114, were incorporated to use as purification tag.
(SEQ ID NO:13)Cln114 Construct: Met1-Ile323(His)6NH2 / MAYVPAPGYQPTYNPTLPYYQPIPGGLNVGMSVYIQGVASEHMKRFFVNFVVGQDPGSDVAFHFNPRFDGWDKVVFNTLQGGKWGSEERKRSMPFKKGAAFELVFIVLAEHYKVVVNGNPFYEYGHRLPLQMVTHLQVDGDLQLQSINFIGGQPLRPQGPPMMPPYPGPGHCHQQLNSLPTMEGPPTFNPPVPYFGRLQGGLTARRTIIIKGYVPPTGKSFAINFKVGSSGDIALHINPRMGNGTVVRNSLLNGSWGSEEKKITHNPFGPGQFFDLSIRCGLDRFKVYANGQHLFDFAHRLSAFQRVDTLEIQGDVTLSYVQIHHHHHH / COOH
A high level of Cln114 expression was observed when the plasmid construct was transformed in E. co...
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