Basic HTML Version
Journal of Mosquito Research, 2013, Vol.3, No.4, 21
-
32
ISSN 1927-646X
http://jmr.sophiapublisher.com
25
transcribed to corresponding cDNA (Figure 3). This
was done using reverse transcriptase enzyme. After
synthesizing the first strand cDNA, it was amplified
using PCR. The PCR product was analyzed by
agarose gel electrophoresis as shown in Figure 4. This
result shows that a single specific PCR product was
obtained with a molecular size of approximately 858
bp from 4
th
instar mosquito larvae that were treated
with entomopathogenic nematodes (
H. bacteriophora
and
S. carpocapsae
) for a period of 3, 6, 9, and 12 hr
but with difference in band intensity between them as
compared to the control (Figure 4a and b).
Semiquantitative determination of the transferrin
transcript abundance revealed that transferrin
expression can be detected in both control and
infected larvae with difference in expression level.
This was clear from the up-regulation of transferrin
gene in case of mosquito larvae infected with
Steinernema nematodes after 3, 6, 9, and 12 hr. This
was obvious from the increased band intensity of
these treated insects in comparison to control group.
Figure 3 Ethidium bromide stained agarose gel
Note: Lane 1 showing cDNA formed from reverse transcription
of isolated total RNA from control
Cx. quinquefasciatus
4
th
instar larvae using RevertAid TM H Minus First Strand cDNA
synthesis kit (Fermentas, cat # K1632). Lane M: Molecular size
markers (G5711, 1 kb DNA Ladder, promega#274214)
Table 1 Integrated display value (IDV) of the obtained PCR product using gel documentation system (Alfainnotech gel
documentation)
Black is 0, White is 255
QUERIES
Band
IDV
%
area
AVG
Rf
Mwt
Position
1
69583
6.8
520
134
0.446
1146.74
598
2
62685
6.1
504
124
0.442
1172.95
596
3
47903
4.7
462
104
0.446
1146.74
598
4
56034
5.5
492
114
0.446
1146.74
598
5
103903
10.1
645
161
0.455
1196.06
602
6
172709
16.8
900
192
0.455
1196.06
602
7
157115
15.3
860
183
0.455
1196.06
602
8
117315
11.4
688
171
0.459
1071.57
604
9
93411
9.1
616
152
0.459
1071.57
604
MARKER
1
145290
14.2
836
174
0.145
10000.00
452
2
-
-
-
-
0.165
8000.00
462
3
-
-
-
-
0.182
6000.00
470
4
-
-
-
-
0.194
5000.00
476
5
-
-
-
-
0.207
4000.00
482
6
-
-
-
-
0.223
3000.00
490
7
-
-
-
-
0.252
2500.00
504
8
-
-
-
-
0.281
2000.00
518
9
-
-
-
-
0.339
1500.00
546
10
-
-
-
-
0.430
1000.00
590
11
-
-
-
-
0.496
750.00
622
12
-
-
-
-
0.612
500.00
678
13
-
-
-
-
0.789
250.00
764