0 using thermal cycling conditions of 15 min at 95°C, followed by 50 cycles of 15 s at 95°C and 1 min at 64°C. A standard curve was generated by plotting the logarithm of the standards copy numbers versus measured C T values. Selleck mTOR inhibitor Isolation of spike-in DNA for use in serial dilutions A crayfish sample extracted from the abdomen of Cherax quadricarinatus (Australian red-claw crayfish) was transferred to
a 2 ml-extraction tube containing 0.7 g Precellys® ceramic beads of 1.4 mm diameter (Peqlab Biotechnology, Erlangen, Germany) and 180 μl buffer ATL, the lysis buffer of the DNeasy® Blood & Tissue Kit (Qiagen). The MagNA Lyser (Roche) was used for three mechanical lysis cycles consisting of 30 s at 6,500 rpm followed by 60 s on a cooling block held at 4°C. Further isolation was performed according to the protocol “”Purification of Total DNA from Animal Tissues (Spin-Column Protocol)”" provided by the manufacturer. DNA concentration was determined
SRT1720 in vitro spectrophotometrically using the Hellma® TrayCell (Hellma, Müllheim/Baden, Germany) on the Eppendorf BioPhotometer 6131. Generation of copy standards A DNA template stock consisting of CHI1, CHI2 and CHI3 sequences was generated as follows. Genomic DNA from Ion Channel Ligand Library screening chitinase sequences were amplified with the primers Chi3-324f20 (5′-TCAAGCAAAAGCAAAAGGCT) and AaChi-Tmr (5′-TCCGTGCTCGCGATGGA). Amplification was evaluated by the signal generated from the TaqMan® probe AaChi-FAM (5′-FAM-TCAACGTCCACCCGCCAATGG-BHQ-1). Amplification was performed in a total volume of 20 μl containing 2 μl 10 × PCR buffer A2 (Solis BioDyne), 0.2 mM of each dNTP, 4 mM MgCl2, 250 nM of each primer, 150 nM TaqMan probe, 1 U HOT FIREPol® DNA polymerase (Solis BioDyne) and 20 ng DNA or water in the case of the no-template control. DNA denaturation and enzyme activation were performed for 15 min at 95°C. DNA was amplified over 50 cycles consisting of 95°C for 15 s, 60°C for 1 min. QPCR was run on StepOnePlus™ Real-Time PCR System (Applied Biosystems) under the StepOne™ software version 2.0. PCR fragments were purified with the MSB® Spin PCRapace Kit (Invitek, Berlin, Germany). The copy number of the target
template was determined spectrophotometrically using Fossariinae the Hellma® TrayCell (Hellma, Müllheim/Baden, Germany) on the Eppendorf BioPhotometer 6131. Serial dilutions of the target sequence (108 to 102, 50, 25 and 12.5 copies per 2 μl) prepared in 10 ng/μl C. quadricarinatus DNA were used to determine the amplification efficiency and the quantitative detection limit. Statistical analysis of expression changes A univariate one-way analysis of variance (ANOVA) with Scheffè’s post-hoc test was used to evaluate the significance of changes in temporal mRNA expression. The dependent variable was the log-transformed mRNA amount. The time was considered a fixed effect. A value of p < 0.05 calculated by the Scheffè’s post-hoc test was regarded as significant.