Biomedical Engineering Reference
In-Depth Information
Table 21.3
Comparison of relative gene expression patterns obtained by microarray
and qRT-PCR. ## Mean of treated to control ratios measured by microarray in three
independent experiments. # Mean of qRT-PCR measurements of the same RNA used in
microarray analyses. Each data point represents the mean
;
D
˙
SE
P
<0:05
0.1 GY
0.5 GY
GENE SYMBOL
Array ##
qRT-PCR #
Array ##
qRT-PCR #
TP53INP1
ND
1:33
˙
0:12
ND
1:738
˙
0:25
1:19
1:236
˙
0:19
1:31
1:43
˙
0:02
CDKN1A
GDF-15
1:33
1:39
˙
0:06
1:44
1:67
˙
0:03
DLGAP4
1:51
1:305
˙
0:13
1:55
1:28
˙
0:10
1:72
˙
0:18
1:58
˙
0:22
KITLG
1:51
1:33
CXCL-12
1:44
0:88
˙
0:079
1:24
1:2
˙
0:2
1:45
˙
0:049
CXCL-2
1:02
1:37
˙
0:29
1:12
RAD54L
1:05
1:06
˙
0:15
0:92
1:01
˙
0:01
(CSNK1D, CRY1, MLH1, TRIB1, ANKRD17, DDIT3, TYMS and POLK) were
up-regulated by 0.1 Gy radiation. Protein biosynthesis and positive regulation of
cell surface receptor linked signal transduction involved genes represented the most
notably up-regulated biological processes after radiation exposure. One of the key
features of the gene expression profile was the high abundance of genes participating
in ubiquitin cascade (UBA52, UBE2D, USP33, UBQLN2 and USP47). The most
significantly down-regulated biological process was regulation of the cell cycle
(Table
21.2
).
QRT-PCR was used to confirm the differential expression of 8 of these genes
(Table
21.3
). Nearly one-third of the genes showed greater fold-changes by qRT-
PCR than predicted by the arrays. Such ratio compression is often encountered in
microarray experiments, and is thought to be probe or primer sequence dependent.
In all, our microarray analysis was well supported by the qRT-PCR results.
21.3.2
Inhibition of GDF-15 gene expression by lentiviral
shRNA constructs
Four lentiviral shRNA constructs were transduced into F11hTERT cells in an at-
tempt to silence the GDF-15 gene. GDF-15 expression was evaluated by qRT-PCR.
Using TRCN#1 (F11hTERT-shGDF-
#
1
/
#
3
)
15
and TRCN#3 (F11hTERT-shGDF-
15
lentiviral constructs relative expression of GDF-15 decreased to
0:448 ˙ 0:058
and
0:59 ˙ 0:106
, respectively (Table
21.3
). Using TRC#2 lentiviral particle construct
(F11-hTERT-shGDF-
2
), the expression of GDF-15 was enhanced instead of
inhibited. There was no significant success in gene silencing effect when we
used the TRC#4 construct. The lentiviral shRNA construct, however, demonstrated
significant advantages in posttranscriptional gene silencing in F11-hTERT-shGDF-
15
#
15
#
1
cells.
