Ultrapure total rna rapid extraction kit

RNA was extracted from the renal tissues using Ultrapure total RNA rapid extraction kit (Bioteke, RP1201, Beijing, China). RNA samples were reverse-transcribed using 5× All-in-One RT MasterMix (Applied Biological Materials Inc., G485&G486&490, Vancouver, Canada) in a 10 µL volume. Next, we performed real-time PCR using EvaGreen 2X qPCR MasterMix- Low ROX (Applied Biological Materials Inc., Vancouver, Canada) in a 20 µL reaction volume. Primers used for quantification were Beclin 1 forward:5′-TGCAGGTGAGCTTCGTGTG-3′ and reverse:5′-CTGGGCTGTGGTAAGTAATGGAG-3′; GAPDH forward:5′-CAAGTTCAACGGCACAGTCAA-3′ and reverse:5′- TGGTGAAGACGCCAGTAGACTC-3′. Relative mRNA levels were determined using the comparative threshold cycle method followed by normalization to the GAPDH mRNA level. Each experiment was performed in duplicates and repeated three times. The used real-time PCR protocol was: 95 °C for 10 min, 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min, 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s.

Total RNA was isolated from the heads of individuals from the stages of early embryo to the adult moth using an Ultra-pure total RNA rapid extraction kit (BioTeke Corporation, CN) according to the manufacturer’s instructions. One microgram of total RNA was used to synthesize cDNA, using the PrimeScript RT reagent Kit with gDNA Eraser (Takara Biomedical Technology, Beijing, CN).