Optimization of Cell-Based cDNA Microarray Conditions for Gene Functional Studies in HEK293 Cells.
Since the cell-based cDNA microarray (CBCM) technique has been a useful tool for gain-of-function studies, many investigators have used CBCMs to identify interesting genes. However, this method requires better-established conditions to ensure high reverse transfection efficiency without cross-contamination. Therefore, we optimized CBCM techniques through various means. We determined that Lipofectamine 2000 was the most appropriate transfection reagent by evaluating eight commercialized reagents, and we determined that the most effective concentrations for printing solution constituents were 0.2 M sucrose (to yield a final concentration of 32 mM) and 0.2% gelatin (to yield a final concentration 0.075%). After examining various combinations, we also determined that the best concentrations of cDNA and transfection reagent for optimal reverse transfection efficiency were 1.5 µg/5 µL of cDNA and 5.5 µL of Lipofectamine 2000. Finally, via a time course, we determined that 72 h was the most effective reaction duration for reverse transfection, and we confirmed the stability of cDNA spot activity of CBCMs for various storage periods. In summary, the CBCM conditions that we have identified can provide more effective outcomes for cDNA reverse transfection on microarrays.
1 Institut Pasteur Korea, Gyeonggi-do, Republic of Korea.|2 Department of Animal Biotechnology and Incurable Disease Animal Model & Stem Cell Institute (IDASI), Konkuk University, Seoul, Republic of Korea.
Hi Chul
HC
Kim
1 Institut Pasteur Korea, Gyeonggi-do, Republic of Korea.
Jin Yeong
JY
Heo
1 Institut Pasteur Korea, Gyeonggi-do, Republic of Korea.
Tae-Kyu
TK
Lee
2 Department of Animal Biotechnology and Incurable Disease Animal Model & Stem Cell Institute (IDASI), Konkuk University, Seoul, Republic of Korea.
Ssang-Goo
SG
Cho
1 Institut Pasteur Korea, Gyeonggi-do, Republic of Korea.|3 Ksilink, Strasbourg, France.
Yong-Jun
YJ
Kwon
D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005780:Gelatin / Q000032:analysis; D005796:Genes; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007202:Indicators and Reagents; D020411:Oligonucleotide Array Sequence Analysis; D013395:Sucrose / Q000032:analysis; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D007202:Indicators and Reagents; D013395:Sucrose; D005780:Gelatin
cDNA microarray; cell image–based array; image-based screening; reverse transfection