Molecular Characterization and Gene Expression Profiling ... - CUSAT

5.2.4. Haemolymph and other tissues collection
Chapter 5
Haemolymph was collected from the rostral sinus using capillary
tubes (RNase-free) rinsed using pre-cooled anticoagulant solution (RNase
free, 10% sodium citrate, pH 7.0). Tissues including gill, muscle, heart,
hepatopancreas and intestine were dissected out using RNase-free scissors
and forceps. Haemolymph and the collected tissues were suspended in TRI
reagent (Sigma) for total RNA isolation.
5.2.5. Total RNA isolation and Reverse transcription
Total RNA isolation and first strand cDNA synthesis were performed
as described in section 2.2.3. to 2.2.6.
5.2.6. Semi-quantitative RT-PCR analysis of target gene expression
Expression of the target genes when supplemented with different
immunostimulants pre- and post-challenge WSSV was determined by semi-
quantitative RT-PCR analysis using 18S rRNA, β-actin and elongation factor
(ELF) as the internal control (Marone et al., 2001). Haemocyte cDNA was
diluted 5 times and amplified using Taq polymerase. PCR amplifications
were performed for three control genes (18S rRNA, β-actin and ELF); five
AMP genes (ALF, crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-
5) and eight WSSV genes (DNA polymerase, endonuclease, immediate early
gene, latency related gene, protein kinase, ribonucleotide reductase,
thymidine kinase and VP28) in a 25 µl reaction volume as described in
section 4.2.6.
From the four different immunostimulants screened, the best one was
selected based on AMP gene expression, intensity of WSSV infection as
envisaged from the WSSV gene expression and post-challenge survival data.
The best performed immunostimulant group along with control group was
subjected to detailed tissue-wise expression profile analysis. Tissue cDNA
was amplified. PCR amplifications were performed for six AMP genes (ALF,
crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5) and two WSSV
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