To quantify NPF mRNA levels, midguts of 8 to 10 adult female flies (5 to 6 days after eclosion) were dissected for each sample. Total RNA was extracted using RNAiso Plus reagent (TaKaRa). cDNA was prepared with ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). RT-qPCR was performed using THUNDERBIRD NEXT SYBR qPCR Mix (TOYOBO) with a Thermal Cycler Dice TP800 system (TaKaRa). Serial dilutions of a plasmid containing the open reading frame of each gene were used as standards. The amount of target mRNA was normalized to ribosomal protein 49 (rp49) and then relative fold changes were calculated. Primers used in this study are described in table S2. The primers for rp49 and NPF were previously described (20 (link)).
Protocol detail
Quantifying NPF mRNA Levels in Drosophila Midguts
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RNAiso Plus reagent
ReverTra Ace qPCR RT Master Mix
gDNA Remover
THUNDERBIRD NEXT SYBR qPCR Mix
Thermal Cycler Dice TP800 system
Adult female
Cdna
Dilutions
Flies
Gene
Mrna
Plasmid
Primers
Ribosomal protein 49
Corresponding Organization : University of Tsukuba
Other organizations : Kurume University, Gunma University
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Variable analysis
independent variables
- Not explicitly mentioned
NPF mRNA levels
- Midguts of 8 to 10 adult female flies (5 to 6 days after eclosion)
ribosomal protein 49 (rp49 )
- Positive control: Serial dilutions of a plasmid containing the open reading frame of each gene were used as standards.
- Negative control: Not explicitly mentioned.
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