One step sybr green rt pcr reagent kit

RNAi-treated cells were subjected to nutrient-full or starvation conditions for 2 or 4 h and were collected and pelleted at 850 rpm for 5 min. Cell pellets were lysed in 1 ml TRIZOL (Invitrogen), and total RNA was extracted according to the manufacturer’s instructions. RNA was treated with DNase, and QRT-PCR was performed using the One-Step SYBR green RT-PCR reagent kit (Applied Biosystems) on a 7900 Sequence Detection System (Applied Biosystems). Expression levels were calculated using the comparative threshold method with Drosophila rp49 as the reference gene. QRT-PCR primers are as follows: rp49, 5′-ATACAGGCCCAAGATCGTGA-3′ and 5′-GCACTCTGTTGTCGATACCCTT-3′, and SesB, 5′-AAGGATTTCGATGCTGTTGG-3′ and 5′-CTCCTTTGGAATGCGGATAA-3′.

Flies were collected and ground in 1 mL TRIZOL (Ambion, 15596–026) and total RNA was extracted according to the manufacturer's instructions. RNA was treated with DNase, and QRT-PCR was performed using the One-Step SYBR green RT-PCR reagent kit (Applied Biosystems, 4389986) on a 7900 Sequence Detection System (Applied Biosystems, Foster City, CA USA). Expression levels were calculated using the comparative threshold method with Drosophila rp49 as a reference gene. QRT-PCR primers are as follows: rp49, 5’-ATACAGGCCCAAGATCGTGA-3’ and 5’-GCACTCTGTTGTCGATACCCTT-3’, and Dcp-1, 5’-CCGGAGTCTCTTGTGTTGGT-3’ and 5’-GTATTCGCTTGCATATCGTTCC-3’.