understanding mirna turnover: a study of mirna half-life dominic mcdonald massachusetts institute of...
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Understanding miRNA Turnover: A Study of miRNA Half-Life
Dominic McDonaldMassachusetts Institute of Technology
Mentor: Jun Lu, Ph. DTodd Golub Laboratory
Broad Institute of MIT and Harvard
What are miRNAs?
miRNAs are short non-coding RNAs
Post-transcriptional regulators of gene expression
miRNAs regulate diverse biological and disease processes
Approach to Measure miRNA t1/2
Culture Cells in Triplicates
Treat Cells with Actinomycin D
Harvest RNA at 6 time points throughout the day
Measure miRNA and control gene expression levels using Luminex
NextGen and RT-PCR
Cell Lines
Maintained 26 different cancer cell lines.
Due to time constraints only able to collect data from 9 different cancer cell lines: HD-MY-Z, 697, HL60, K562, Ramos (RA1), RS4;11, SEMK2, U937, and HEL cell lines.
Triplicates of every cell line done at 6 time points each for a total of 162 samples.
Control Gene Degradation c-myb
U937 c-myb
0
20
40
60
80
100
120
140
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Time (hr)
% E
xp
res
sio
n
Control Gene Degradation c-myb
U937 c-myb
-19
-18
-17
-16
-15
-14
-13
-12
-11
-10
0 2 4 6 8 10 12 14
Time (hr)
log
2 e
xp
res
sio
n
Control Gene Degradation c-myc
SEMK2 c-myc
-22
-21
-20
-19
-18
-17
-16
-15
-14
-13
0 2 4 6 8 10 12 14
Time (hr)
log
2 e
xpre
ssio
n
RT-PCR Validation
Use additional methodology to validate results
Use RT-PCR for validation Measure miR-125a in K562 cells Measure u6 small RNA as a control
RT-PCR Validation
K562 miR-125a
20
21
22
23
24
25
26
27
28
29
30
0 5 10 15 20 25
Time (hr)
Raw
Exp
ress
ion
Ct
RT-PCR Validation
K562 u6
28
29
30
31
32
33
34
35
36
37
38
0 5 10 15 20 25
Time (hr)
Raw
Exp
ress
ion
Ct
What Does This All Mean?
Most miRNAs have long t1/2 (over 24 hours)
We have not observed any fast degrading miRNAs (t1/2 less than 8 hours)