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