Detection of PRAT satellite transcripts.

<p><i>(A)</i> RT-PCR with primers RATZEB1 and RATZEB2 on total RNA from adults (ad), larvae (lar) and pupae (pup). For each reaction PCR was conducted in parallel without prior reverse transcription as a control for genomic DNA contamination, while 0 is control with no template. M is size standard. <i>(B)</i> Total RNA separated on 1.2% denaturing agarose gel (18 µg and 13 µg from adults and larvae, respectively) and corresponding Northern blot after hybridization with ³²P labelled PRAT DNA probe. 18 µg of adult RNA digested with RNase A represents control for residual genomic DNA (+R) while 18 µg of <i>D. melanogaster</i> RNA (c) is used as a negative control. <i>(C)</i> Dot blot analysis of total RNA, using ³²P labelled RNA probes for forward and reverse strand, respectively. 20 µg and 50 µg of larval RNA digested with RNase A represent control for genomic DNA contamination (+RNase) while the same amounts of <i>S. cerevisiae</i> RNA are used as a negative control (−control). Complementary transcripts, in amounts indicated, are used as a positive control (+control) and for calibration. <i>(D)</i> 30 µg of larval low molecular weight RNA (lar) separated on 12% polyacrylamide gel and Northern blot analysis with RNA probe for reverse strand, labelled with ³²P. Hybridization was performed at 22°C and blot was exposed for one day on autoradiography film. Longer exposition as well as rehybridization with RNA probe for forward strand did not reveal signal in the siRNA region. <i>D. melanogaster</i> RNA (dm) serves as a size marker.