Poly(A)+ RNA was extracted from 100mg of female flies from either Oregon R or Su(H)gwt using the Poly ATract 1000 kit (Promega, Mannheim, Germany), and cDNA generated with ProtoScriptII First Strand cDNA Synthesis Kit (New England Biolabs, Frankfurt, Germany) according to the suppliers’ protocols. The PCR spans the first intron that contains the manipulations: Genomic DNA from wild type and from Su(H)gwt should yield a 1012 bp and a 1745 bp amplificate, respectively. The amplificate from cDNA is expected to be 238 bp in size when spliced normally. The following primer pair was used: Pu, 5’ CCG GCC ACA CAT CGA GGA GAA G 3’ and Pl, 5’ CGC GCA TAG TTG TGC TCC CTG TTC G 3’.
qPCR was done on three biological replicates of each genotype with 40 homozygous larvae at ~72 hours after egg deposition at 25°C. Poly(A)+ RNA was extracted with Poly ATract® 1000 kit (Promega, Mannheim, Germany) and the concentration determined in a μCuvette with the BioPhotometer Plus (Eppendorf, Hamburg, Germany). 1μg was treated with 0.4U DNase I (New England Biolabs, Frankfurt, Germany) and reverse transcribed in 0.3μg batches with the ProtoScriptII Kit using oligo-dT primers (New England Biolabs, Frankfurt, Germany). Real time qPCR was performed with Blue S’Green qPCR Kit (Biozym, Hessisch-Oldendorf, Germany) on 2μl of cDNA (0.012μg) in 10μl end volume using MIC magnetic induction cycler (bms, Pots Point Australia) always including target and no-template controls; a hot start (95°C 2 min) and 40 cycles of 95°C 5s / 68°C 10s was followed by a melt curve analysis (72°C to 95°C at 0.3°/s) to select for specific amplification. Absence of DNA was tested in a non-RT control for every sample; RNA integrity was confirmed by 5’-3’ Cq analysis. CTCF (PP30808), cyp33 (PP14577), DNApol-α60 (PP9936), eRF1 (PP11596), hisRS (PP13550), and Tbp (PP1556) were assayed as internal references; primer pair sequences (in parentheses) are listed at DRSC FlyPrimer bank [65 (link)]. Su(H) primers (Upper, 5’ CAT ATC CAC CGA CAA GGC TGA GTA CC 3’; Lower, 5’ TAA CGA TTG GCA CTG GAG TGA CTG G 3’) span the second intron. Eventually, cyp33 and Tbp were selected based on variance, Cq values, and expression profiles matching that of Su(H) (DRSC FlyPrimer bank). Relative quantification of the data was performed with micPCR software Version 2.2 based on REST [66 (link)], taking target efficiency into account.
qPCR was done on three biological replicates of each genotype with 40 homozygous larvae at ~72 hours after egg deposition at 25°C. Poly(A)+ RNA was extracted with Poly ATract® 1000 kit (Promega, Mannheim, Germany) and the concentration determined in a μCuvette with the BioPhotometer Plus (Eppendorf, Hamburg, Germany). 1μg was treated with 0.4U DNase I (New England Biolabs, Frankfurt, Germany) and reverse transcribed in 0.3μg batches with the ProtoScriptII Kit using oligo-dT primers (New England Biolabs, Frankfurt, Germany). Real time qPCR was performed with Blue S’Green qPCR Kit (Biozym, Hessisch-Oldendorf, Germany) on 2μl of cDNA (0.012μg) in 10μl end volume using MIC magnetic induction cycler (bms, Pots Point Australia) always including target and no-template controls; a hot start (95°C 2 min) and 40 cycles of 95°C 5s / 68°C 10s was followed by a melt curve analysis (72°C to 95°C at 0.3°/s) to select for specific amplification. Absence of DNA was tested in a non-RT control for every sample; RNA integrity was confirmed by 5’-3’ Cq analysis. CTCF (PP30808), cyp33 (PP14577), DNApol-α60 (PP9936), eRF1 (PP11596), hisRS (PP13550), and Tbp (PP1556) were assayed as internal references; primer pair sequences (in parentheses) are listed at DRSC FlyPrimer bank [65 (link)]. Su(H) primers (Upper, 5’ CAT ATC CAC CGA CAA GGC TGA GTA CC 3’; Lower, 5’ TAA CGA TTG GCA CTG GAG TGA CTG G 3’) span the second intron. Eventually, cyp33 and Tbp were selected based on variance, Cq values, and expression profiles matching that of Su(H) (DRSC FlyPrimer bank). Relative quantification of the data was performed with micPCR software Version 2.2 based on REST [66 (link)], taking target efficiency into account.
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