Murine leukaemia virus reverse transcriptase

Blood was obtained from infected mice exhibiting 2–5% parasitaemia. Total RNA was isolated from blood containing 5 × 10⁶ or 1 × 10⁷ infected erythrocytes using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. Total RNA was then treated with DNase and reverse-transcribed using murine leukaemia virus reverse transcriptase (Applied Biosystems, Carlsbad, CA, USA) with random hexamer primers under the following conditions: 70 °C for 10 min, 25 °C for 10 min, and 42 °C for 30 min. The reaction was terminated by heating at 99 °C for 5 min, and the resulting cDNA products were stored at −20 °C until required. All PCR reactions were run in a 25 μL volume consisting of 1×TaKaRa Ex Taq buffer, 2.5 mM dNTP, 1 μL of cDNA products, 5 U/μL TaKaRa Ex Taq DNA polymerase, and PCR primers (0.25 μM); a list of primers used for semi-quantitative RT-PCR can be found in Additional file 2. PCR reactions were performed on a C1000 thermal cycler (Bio Rad) for 30 cycles under the following conditions: denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 45 s. Products were resolved on a 2% (w/v) agarose gel, and stained with ethidium bromide. Samples with DNase-treated RNA template were used as the negative control.

For total RNA isolation, the SV Total RNA Isolation System with on-column DNase digestion (Promega, Madison, WI, USA) was used. The first-strand cDNA synthesis was carried out with DNase-treated RNA (200 ng) by using Murine Leukaemia Virus Reverse Transcriptase and Oligo d(T)16 (Applied Biosystems, Forster City, CA, USA). One-tenth of the cDNA reaction was taken as PCR template and amplified for 25–35 cycles depending on the relative mRNA quantity with denaturation at 94 °C for 15 s, annealing at 58 °C to 64 °C for 15 s according to the primers, and elongation at 72 °C for 30 s. The forward and reverse primer sequences, the annealing temperatures, and the number of cycles being performed for RT-PCR analyses are listed in Table S3. GAPDH was used as an internal control.