One step real time pcr protocol

The animals fed on aex-5 RNAi plates or treated with S. aureus or arecoline following the above-described protocol were collected, washed with M9 buffer, and frozen in QIAzol reagent (Qiagen, Netherlands). Total RNA was extracted using the RNeasy Plus Universal kit (Qiagen, Netherlands). A total of 6 μg of total RNA was reverse transcribed with random primers using the High-Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA).
Quantitative reverse transcription-PCR (qRT-PCR) was conducted using the Applied Biosystems one-step real-time PCR protocol using SYBR green fluorescence (Applied Biosystems) on an Applied Biosystems 7900HT real-time PCR machine in 96-well plate format. Twenty-five-microliter reaction mixtures were analyzed as outlined by the manufacturer (Applied Biosystems). The relative fold changes of the transcripts were calculated using the comparative cycle threshold (C_T) (2^−ΔΔCT) method and normalized to pan-actin (act-1, act-3, and act-4). The cycle thresholds of the amplification were determined using StepOnePlus software (Applied Biosystems). All samples were run in triplicate. The primer sequences are listed in Table S3.

Animals were synchronized and total RNA extraction was done following the protocol described above. qRT-PCR was conducted using the Applied Biosystems One-Step Real-time PCR protocol using SYBR Green fluorescence (Applied Biosystems) on an Applied Biosystems 7900HT real-time PCR machine in 96-well-plate format. Twenty-five-microliter reactions were analyzed as outlined by the manufacturer (Applied Biosystems). The relative fold-changes of the transcripts were calculated using the comparative CT(2^−ΔΔCT) method and normalized to pan-actin (act-1, −3, −4). The cycle thresholds of the amplification were determined using StepOnePlus Real-Time PCR System Software v2.3 (Applied Biosystems). All samples were run in triplicate. The primer sequences were available upon request and presented in Table S8.