[Establishment of the pipeline for RNA quality assessment from the cells obtained by laser capture microdissection].
We developed a standard protocol for quality assessment of low amount RNA from the cells obtained by laser capture microdissection (LCM). Three gastric noncancerous tissues were cryo-sectioned, stained with Cresyl Violet, and pathologically rechecked. Epithelial cells were obtained by LCM and RNA was isolated. Agilent 2100 bioanalyzer was used to check the RNA quality. To validate the results from 2100 bioanalyzer, RT-PCR was performed with six genes at both 5'and 3' end-regions of different abundance (EF1A and ATCB of high abundance, GAPDH and B2M of moderate abundance, and MED1 and CK20 of low abundance). RT-PCR analysis of 3 good quality RNAs from cultured cell lines and 3 poor quality RNAs from gastric noncancerous tissues showed high correlations with that from 2100 bioanalyzer. In conclusion, the pipeline for low amount RNA quality assessment by RT-PCR from tissue cryo-section, pathological recheck, LCM purification and RNA isolation is applicable as a routine method in cancer genome research.
Yang
Yan-Qing
YQ
State Key Laboratory of Medical Genomics and Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. yanqing_yang@shbiochip.com
Zhang
Wen
W
Zhang
Bao-Feng
BF
Gao
Heng-Jun
HJ
Zhang
Qing-Hua
QH
D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary; D004847:Epithelial Cells / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007834:Lasers; D042282:Microdissection; D011786:Quality Control; D012313:RNA / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification / Q000592:standards*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics / Q000473:pathology
D012313:RNA