Q–PCR analysis detects transcription in pre-blastoderm embryos.

<p>(A) RNA extracted from wildtype embryos at the indicated cycles, from unfertilized eggs (0) and from <i>Df(en)</i> cycle 14 – germ band extended embryos (GBE) was used to template PCR using primers that nestle <i>en</i> intron 1 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003428#pgen.1003428.s002" target="_blank">Table S1</a>). No product was detected after 40 PCR cycles using RNA extracted from unfertilized eggs or from <i>Df(en)</i> cycle 10–12 embryos. Product using primers for actin transcripts was detected in all samples. (B) cDNAs prepared from RNA extracted from wildtype embryos at the indicated nuclear cycles was used to template PCR using primers that nestle an intron of <i>en, h, odd, Kr</i>, and <i>eve</i>. Amount of RNA used to prepare the cDNA templates for PCR amplification: (A) 30 ng for nuclear cycles 0–9; 5 ng for nuclear cycles 9–14 and GBE; (B) 30 ng for nuclear cycles 3–6 and 8. PCR reactions were performed in an ABI 3730 Thermocycler for (A) and a BioRad C1000 Touch Thermal Cycler for (B); the number of PCR cycles required to amplify <i>en</i> transcripts differed with the two instruments (presumably due to distinct cycling protocols), but was reproducible for each instrument.