By: Carol Tran Dr. Zendra Zehner Dr. Xueping Zhang
By: Carol Tran
Dr. Zendra Zehner
Dr. Xueping Zhang
MicroRNA Review
http://www.jyi.org/articleimages/392/originals/img2.jpg
To test if miR23 and/or miR17 have an effect on the growth, tumorigenic, or
metastatic properties of prostate cancer cells.
Prostate Gland Cross-SectionF6
M12
0
5
10
15
20
25
30
M12 F6
rela
tive
miR
23-L
uc a
ctiv
ity
miR23 on Chromosome 19
p69 (prostate epithelial subline)
0
10
20
30
40
50
M12 F6
Am
ount
Pre
sent
p69 (prostateepithelialsubline)
Summer Goal1.) To monitor the levels of the microRNAs in
M12 compared to the F6 subline.
2.) To change the levels of the microRNA and measure the
differences in the reporter gene activity.
http://www.clontech.com/products/detail.asp?product_id=10606&tabno=2
5’ 3’
Process• Clone DNA
• Transform Bacterial Cells to take up Plasmid DNA molecule
• Mini-Preps and Maxi-Preps
• Monitor Effect on Reporter Gene Activity- Transfection
• Assays for Results
hsa-miR-23a: ATCACATTGCCAGGGATTTCC
5’GATCCGATCACATTGCCAGGGATTTCCTTTTCAAGAGAGGAAATCCCTGGCAATGTGATTTCTTTTTTTCTAGAG gCTAGTGTAAGGTCCCTAAAGGAAAAGTTCTCTCCTTTAGGGACGTTACACTAAAGAAAAAAAGATCTCTTAA 5’
hsa-miR-23a-AS: GGAAATCCCTGGCAATGTGAT
5’GATCCGGGAAATCCCTGGCAATGTGATTTTTCAAGAGAATCACATTGCCAGGGATTTCCTTCTTTTTTTCTAGAG gCCCTTTAGGGACCGTTACACTAAAAAGTTCTCTTTAGTGTAACGGTCCCTAAAGGAAGAAAAAAAGATCTCTTAA 5’
0.0
0.5
1.0
1.5
pCAT3 pCAT3-Vim-3'UTR
CA
T A
ctiv
ity n
orm
aliz
ed to
pC
AT3 M12
F6
miR17 miR17
0
5000
10000
15000
20000
25000
0ug 0.05ug 0.1ug 0.3ug 1ug
Amount mir17 plasmid
CA
T A
ctiv
ity
miR17
0.00
0.50
1.00
1.50
2.00
2.50
3.00
M12 M12+miR23-As
Rel
ativ
e Lu
cife
rase
Act
ivity
mir23
Future Ideas• Could measure exact amount of miRs in M12 and F6 sublines instead of reporter gene activity.
• Northern Blot and radioactivity
CONCLUSIONCONCLUSION
o Poured plates o Mini plasmid preps o Maxi plasmid preps o Took ODs o dual-Luciferase Assayo Culture Cellso Transfections