Construction of cDNA library of osteosarcoma cell OS-9607 and its quality analysis
- VernacularTitle:构建骨肉瘤OS-9607细胞cDNA文库及其质量分析
- Author:
Dawei HE
;
Jun LIU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2006;10(44):212-214
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Osteosarcoma cell OS-9607 is a cell line cultured from human osteosarcoma tissue. To construct cDNA library of this cell line so as to clone target gene from it.OBJECTIVE: To construct cDNA library of osteosarcoma cell OS-9607 by switching mechanism at 5' end of RNA transcript (SMART) technique and analyze its quality.DESIGN: Verified experimental study SETTING: Department of Orthopaedics, Changhai Hospital, Second Military Medical University of Chinese PLA MATERIALS: This experiment was carried out at the Department of Orthopaedics, Changhai Hospital, Second Military Medical University of Chinese PLA in May 2005. Osteosarcoma cell OS-9607 was cultured in CO2 incubator. Cells were pre-treated with DM for 30 minutes, recovered for 24 hours in standard culture medium. The whole RNA of osteosarcoma cell OS-9607 was isolated by RNAease reagent kit. The quantity and integrity of total RNA was detected by ultraviolet spectrometer and electrophoresis on a denaturing formaldehyde agarose gel stained by ethidium bromide (EtBr). METHODS: Total RNA was extracted from human osteosarcoma cell OS9607 and mRNA was isolated, cDNA of OS-9607 cells was acquired by in situ polymerase chain reaction (PCR) and long-distance PCR (LD-PCR),then the cDNA was ligated with dephosphorylated λ phage vector. The recombinants were constructed with SMART cDNA library construction kit.The size of cDNA inserts and the diversity of library were detected by PCR.MAIN OUTCOME MEASURES: The quality and integrity of total RNA, the titer of un-amplified and amplified library and recombination fraction; analysis of cDNA length of library RESULTS: ①The concentration of the isolated total RNA was determined and the measuring absorbance was between 260 nm and 280 nm. Its absorptance was about 1.9 A. 0.8％ denaturing formaldehyde agarose gel electrophorosis showed clear 28S rRNA and 18S rRNA,and the brightness rate of 28S rRNA and 18S rRNA (28S/18S) was 2:1. ②After linked to λ phage in vitro, packed and transfected host bacteria, the titer of unamplified library was 2.2×107 pfu/mL, and the titer of amplified 1 ibrary arrived to 4.5×1010 pfu/mL. The recombination fraction analysis showed that there were 8 blue plaques and 1 360 colorless plaques in one plate, so the recombination fraction was 99.5％. ③ The obtained cDNA library consisted of 1.6×106 recombinant bacteriophages and the average exogenous inserts of the recombinants was 1.5 kb.CONCLUSION: These results suggest that the obtained osteosarcoma cell OS-9607 cDNA library has high quality, which sets up a firm foundation for the further research on osteosarcoma cell OS-9607 and its elated genes screen.
