Rnaqueous 4pcr dna free rna isolation for rt pcr kit

For RT-PCR analysis, fifteen 0- to 3-d-old yw females were cultured overnight on standard medium supplemented with wet yeast paste. Head, ovaries, and carcasses were dissected in RNAlater (Ambion) for analysis of endogenous AdipoR isoforms. For qPCR analysis, fifty 0–1 day old yw females were cultured for one week on standard medium supplemented with wet yeast (“rich diet”) or molasses/agar medium (“poor diet”), then dissected in RNAlater. To avoid potential contributions of stage-specific differences in AdipoR expression, vitellogenic egg chambers (which are differentially represented on rich versus poor diets) were removed from ovaries prior to RNA extraction. RNA was extracted from all tissues using RNAqueous-4PCR DNA-free RNA Isolation for RT-PCR kit (Ambion) according to the manufacturer’s instructions. cDNA was synthesized using SSRII kit (Ambion) according to manufacturer’s instructions and used immediately for PCR using primers listed in Table S1 in Supplementary material. Rp49 was used as a control for RT-PCR. For qPCR, reactions were performed with SYBER Green Supermix (Bio-Rad), and Rp49, Actin 5C, and α-Tubulin were used as controls.

For RNA extraction, 5 whole females, 5 pairs of ovaries, 10 abdominal carcasses (including adipocytes and oenocytes, but without ovaries and gut), 10 guts, 10 thoraces, or 10 heads were incubated in RNAlater (Ambion) for 30 min according to the manufacturer’s instructions to stabilize RNA. Samples were then incubated in 250 μl lysis buffer from the RNAqueous-4PCR DNA-free RNA isolation for RT-PCR kit (Ambion). RNA was extracted from all samples using a motorized pestle and following the manufacturer’s instructions. The primers used for the PCR reactions are listed in Supplemental Material, Table S1. Rp49 primers were used as a control. Net band intensity for each sample was quantified using ImageJ by subtracting background pixels from band pixels in a fixed-size box, and normalized to the band intensity for the corresponding Rp49 band. Controls were set to one and experimental sample intensities were calculated relative to control.