Total RNA was prepared from PAG using Trizol reagent (Invitrogen,USA), and further purified with an QIAGEN RNeasy Kit
Label
Cy3
Label protocol
Total RNA were primed with T7 Promoter Primer (from the Agilent Low RNA Input Linear Amplification Kit PLUS, Two-Color) at 65? for 10 min, then reversed transcribed at 40? for 2 h in the presence of 1ul MMLV-RT (Agilent), and 10 mM dNTP, and RNase OUT (Agilent) and labeled with either Cy3-dCTP or Cy5-dCTP. Equal amounts of the RNA from control sample were mixed, and the mixture was labeled with Cy5, while the RNA from each PAG which come from rats received 2Hz electroacupuncture for 30 min, nociceptive testing, returned to home cages for 24 hours and non-sensitivity on electroacupuncture analgesia was labeled with Cy3.
strain: Sprague-Dawley treatment: control tissue: PAG
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from PAG of control rats using Trizol reagent (Invitrogen,USA), and further purified with an QIAGEN RNeasy Kit
Label
Cy5
Label protocol
Total RNA were primed with T7 Promoter Primer (from the Agilent Low RNA Input Linear Amplification Kit PLUS, Two-Color) at 65? for 10 min, then reversed transcribed at 40? for 2 h in the presence of 1ul MMLV-RT (Agilent), and 10 mM dNTP, and RNase OUT (Agilent) and labeled with either Cy3-dCTP or Cy5-dCTP. Equal amounts of the RNA from control sample were mixed, and the mixture was labeled with Cy5, while the RNA from each PAG which come from rats received 2Hz electroacupuncture for 30 min, nociceptive testing, returned to home cages for 24 hours and non-sensitivity on electroacupuncture analgesia was labeled with Cy3.
Hybridization protocol
After mixed with hybridization buffer(GE healthcare), samples were applied to microarray slide, cover the slip with care. Place the slide in a wet box.Incubate in a hybridization chamber at 42? for 16-18 hours, avoid light exposure. After hybridization, slides were washed sequentially with 1 SSC/0.2 SDS(50°C), 0.1 SSC/0.2%SDS(50°C) and 0.1 SSC(room temperature) before scanning.
Scan protocol
Axon GenePix 4000B
Description
Channel 1: Sprague-Dawley rats were received 2Hz electroacupuncture for 30 min, nociceptive testing and returned to home cages for 24 hours before sacrificed.These rats were non-sensitivity on EA analgesia. It's PAG was then collected for transcript profiling analysis. Channel 2: strain: Sprague-Dawley rats were sacrificed after stayed in their home cages without receiving electroacupuncture stimulation.the RNA of PAG were collected for transcript profilinf analysis Total RNA was prepared by using TRIzol reagent (Life Technologies, USA), further purified with an RNEasy column (Qiagen, Germany), and quantified by using gel electrophoresis and spectrophotometer followed by reverse transcription labeling. Each RNA sample was reverse-transcribed into cDNA primed with oligo(dT) and labeled with either Cy3-dCTP or Cy5-dCTP by using Superscript II reverse transcriptase (Life Technologies, USA). After hybridization under a standard protocol, data acquisition was performed by using a laser scanner (Agilent, USA), and the image data were analyzed using ImaGene 4.2 (Biodiscovery, Santa Monica, CA, USA).
Data processing
LOWESS normalized, background subtracted VALUE data obtained from log of pro cessed Red signal/processed Green signal
