Illustra rnaspin mini isolation kit

Using the Illustra RNAspin Mini isolation kit (GE Healthcare 25-0500-70), total mRNA was extracted from pools of 10 zebrafish embryos at 48 hpf, from pools of 5 chick embryos at HH11 stage or from mouse embryos at stage 8.5 dpc. Reverse transcription of total RNA was performed using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific), according to the manufacturer’s guidelines. RT-qPCRs were performed in a Step One Plus machine (Applied Biosystems) using Fast SYBR Green Mastermix (Applied Biosystems). RNA expression levels were calculated using the comparative Ct method normalized to the internal control genes indicated below. The final results were expressed as the relative RNA levels, calculated with the 2^-(ΔΔCt) method. Data are represented as the mean±s.e.m. of at least three independent experiments with each point analysed in triplicate. The primers used are listed in Supplementary Table II.

Total RNA extraction, conversion into complementary DNA and qRT-PCR were performed as previously described (Browning and Karim 2013 (link)). Briefly, the tick midguts and salivary gland tissues stored in RNAlater were used for total RNA extraction using Illustra™ RNAspin Mini Isolation Kit (GE Healthcare, Piscataway, NJ, USA) according to manufacturer’s instructions. Total RNA (~ 1 µg) was reverse transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase according to the manufacturer’s protocol (Invitrogen). Gene-specific primers were designed to amplify the cDNA fragments from A. maculatum tissues. All primer sequences used in this study are listed in Table 1. First-strand cDNA was used to measure mRNA levels using qRT-PCR. Maxima™ SYBR Green qPCR Master Mix (2×) (Fermentas Life Sciences, Waltham, MA, USA). cDNA (25 ng) and 150 nM of gene-specific primers (Table 1) were used in each reaction mixture (Browning et al. 2013 (link)). Reaction mixtures were subjected to 95°C for 10 min, followed by 35 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s using the CFX96 Real Time System (BIORAD Inc.). All samples were run in triplicate and Bio-Rad Software was used for data analysis with the 2^ddCt method.