[Expressed genes related to adolescent idiopathic scoliosis in spinal facet].
OBJECTIVE
To construct a subtractive cDNA library from the spinal facets of adolescent idiopathic scoliosis (AIS) patient with suppression subtractive hybridization (SSH) technique and clone differentially expressed genes related to AIS in spinal facet.


METHODS
mRNA was isolated separately from the convex side and concave side apical facets of an adolescent idiopathic scoliosis patient. Moreover, single-strand (ss) and double-strand (ds) cDNAs were synthesized in turn using SMART PCR cDNA synthesis technology. (ds) cDNAs then were digested with Rsa I and divided into two groups, and ligated to the specific adaptor 1 and adaptor 2R, respectively. After cDNAs were hybridized with each other twice and underwent two rounds of nested PCR, the PCR products were ligated with pDrive Cloning Vectors to set up the subtractive library. Sequence analysis was performed and the acquired data were aligned against the GeneBank nucleotide database. Furthermore, the spinal facets of 11 IS patients were collected. The techniques of immunohistochemistry and in situ hybridization were adopted in order to approve the gene expression difference. The images of immunohistochemistry and in situ hybridization were input to the image analysis system and were analyzed semi-quantitatively.


RESULTS
A cDNA subtractive library of AIS in spinal facet was set up successfully with high subtractive efficiency. Beta2 Microglobulin (beta 2M) and calgranulin A (S100A8) were identified as highly differentially expressed genes in this study.


CONCLUSIONS
All results confirm the effectiveness and sensitivity of SSH in detecting differentially expressed genes from a small amount of clinical samples. Information about such alterations in gene expression can be useful for elucidating the genetic events in the development of AIS.
Qiu
Gui-xing
GX
Orthopedic Department of Peking Union Medical College Hospital, CAMS and PUMC, Beijing 100730, China.
Qian
Wen-wei
WW
Wu
Zhi-hong
ZH
Liu
Yong
Y
Xiao
Jun
J
Wang
Yi-peng
YP
Zhang
Jian-guo
JG
Shen
Jian-xiong
JX
Weng
Xi-sheng
XS
Zhao
Zhen-guo
ZG
D000293:Adolescent; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008297:Male; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012600:Scoliosis / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D021801:Zygapophyseal Joint / Q000378:metabolism
D012333:RNA, Messenger