||Fruit fly Drosophila melanogaster
||Thummel CS, Burtis KC, Hogness DS. Spatial and temporal patterns of E74 transcription during Drosophila development. Cell. 1990 Apr 6 61(1):101-11. abstract p.102 left column 3rd paragraph & p.103 left column 2nd paragraph & p.108 left column 2nd paragraphPubMed ID1690603
||"Six 150 mm petri dishes containing mass-isolated larval organs were
maintained at 25°C in Robb's saline as described above. These were
incubated for 2 hr to allow recovery from any prior exposure to ecdysone
in vivo. Ecdysone was then added to each plate at timed intervals.
At the end of the time course the organs were chilled and collected by
a 30 s centrifugation at 1500 rpm. The organs were disrupted in 5 ml
of cold lysis buffer (10 mM Tris [pH 7.8], 150 mM NaCl, 0.65% NP-40)
by 5 strokes with an A pestle in a glass dounce homogenizer. The nuclei
were pelleted by centrifugatfon at 7000 rpm for 2 min at 5°C. The supernatant
cytoplasm was poured into phenol, and the nuclear pellet was
resuspended in 6 M guanidine-HCl, 0.1 M sodium acetate (pH 5.2) and
treated as described above for RNA extraction. The cytoplasmic RNA
was purified by phenol extraction, ether extraction, and ethanol
precipitation. These samples were resuspended in 30 mM Tris (pH 7.8)
10 mM MgCl2, and 3.5 mg of RNAase-free DNAase (Worthington) was
added. After incubation at 37°C for 30 min the RNA was purified by
phenol extraction, ether extraction, and ethanol precipitation. Nuclear
and cytoplasmic RNAs were denatured with formaldehyde as described
above and loaded onto Nytran using a plexiglass slot blot apparatus.
The Nytran strips were hybridized with single-stranded radioactive
DNA probes as described above for Northern blots."
||"Densitometric scanning and normalization of the data
obtained with the E74A specific probes yielded an E74A
transcription elongation rate of 1.1 kb/min at 25°C, which
[researchers] estimate is accurate to ±30%. This result is consistent
with in vivo transcription rates reported by others (Kafatos,
1972: Suzuki, personal communication: Ucker and Yamamoto,
1964). Figure 1 also shows that cytoplasmic E74A
mRNA was not detected until 60 min after the addition of ecdysone."