Preparation of novel magnetic cellulose microspheres via cellulose binding domain-streptavidin linkage and use for mRNA isolation from eukaryotic cells and tissues.
We have developed micron-sized magnetic cellulose microspheres (MCMS), biospecifically coated with streptavidin (SA) using a novel cellulose binding domain and SA fusion protein, to harvest mRNA from eukaryotic cells and tissues. Biospecific connection between SA and MCMS exhibited significant advantages compared with traditional chemical coupling, including convenient and simple preparation, elimination of toxic compounds, and highly efficient extraction of a pure target molecule. To confirm compatibility, mRNA isolated from cell or tissue samples was used for amplification of housekeeping genes (GAPDH and beta-actin) and specific target genes (two fragments V(H) and V(L) from the variable fragments of IgG and the gene encoding GLP-1 receptor). Preliminary studies predict that this novel method has potential in future developments in target gene isolation and DNA manipulation.
Gao
Zhihui
Z
Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China.
Zhang
Qi
Q
Cao
Yu
Y
Pan
Pengwei
P
Bai
Fang
F
Bai
Gang
G
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D005057:Eukaryotic Cells / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D005821:Genetic Techniques; D008280:Magnetics; D008863:Microspheres; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C074074:cellulose binding protein A, Clostridium cellulovorans; D019809:Streptavidin