Total RNA was isolated as described previously. First, 0.5 μg total RNA of each sample was transcribed into complementary deoxyribonucleic acid (DNA) by using avian mycoblastosis virus reverse transcriptase (AMV RT) and random hexamer primers according to the supplier's instructions (Roche Applied Sciences, Mannheim, Germany). Then, 3 μl of each complementary DNA preparation was diluted to a final PCR volume of 25 μl containing 12.5 μl TaqMan Universal Master Mix (Applied Biosystems, Foster City, California), and 0.25 μl (200 nM) primers (TIB Molbiol, Berlin, Germany) and 0.3 μl (100 nM) probes as listed in (Table 1). Each sample was tested for the following mRNAs: TF, asHTF, and GAPDH. In order to differentiate between TF and asHTF, primers and probe for detection of TF have been positioned in exon 5, which is missing in asHTF, whereas asHTF primers have been positioned in exon 4 and 6, respectively, with the probe spanning exon 4/6 boundary, which is not present in TF. Real-time PCR was performed using ABI Prism 7000 Sequence Detection System (Applied Biosystems) under the following conditions: 50°C, 2 min; 95°C, 10 min; 40 cycles 95°C, 15 s, 60°C, 1 min. Standards covering the complete coding sequence for each target were generated by RT-PCR. Serial dilutions of each standard were made.