Strain: Fischer 344, Gender: Male, Age: 8-9 weeks, Exposure: inhalation of 500 ppm of COS, 6 hours per day for 2 consecutive days
Extracted molecule
total RNA
Extraction protocol
Total RNAs from frozen brain tissue (posterior colliculus) were isolated with an RNeasy tissue kit (Qiagen, Valencia, CA) according to the manufacturer's protocols. The quality and integrity of the RNA was verified by 260/280 nm absorbance ratio, formaldehyde agarose gel electrophoresis and an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Label
Cy3
Label protocol
Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy3 labeled cRNA was produced according to manufacturer’s protocol.
Total RNAs from frozen brain tissue (posterior colliculus) were isolated with an RNeasy tissue kit (Qiagen, Valencia, CA) according to the manufacturer's protocols. The quality and integrity of the RNA was verified by 260/280 nm absorbance ratio, formaldehyde agarose gel electrophoresis and an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Label
Cy5
Label protocol
Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy5 labeled cRNA was produced according to manufacturer’s protocol.
Hybridization protocol
For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol.
Scan protocol
Slides were washed as indicated in the Agilent 60-mer oligo microarray processing protocol and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software, using defaults for all parameters.
Description
Immediately after the cessation of COS exposure on the appropriate day of sacrifice (day 1 or day 2) the rats were deeply anesthetized with intraperitoneal sodium pentobarbital. The posterior colliculi were surgically exposed and removed using a pair of corneal scissors and fine forceps. The samples were immediately placed in RNase-free plastic vials and snap frozen in liquid nitrogen. Three control animals (non-exposed) were processed identically. Total RNAs from frozen brain tissue (posterior colliculus) were isolated with an RNeasy tissue kit (Qiagen, Valencia, CA) according to the manufacturer's protocols. Gene expression analysis was conducted using Agilent Rat Oligo Microarrays (Agilent Technologies, Palo Alto, CA). Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy3 or Cy5 labeled cRNA was produced according to manufacturer’s protocol. For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Two arrays were utilized for each comparison allowing for dye reversals. Data was obtained using the Agilent Feature Extraction software (v8.1), using defaults for all parameters and loaded into the Rosetta Resolver® system (v6.0) (Rosetta Biosoftware, Kirkland, WA).
Data processing
Data was obtained using the Agilent Feature Extraction software (v8.1), using defaults for all parameters and loaded into the Rosetta Resolver® system (v6.0) (Rosetta Biosoftware, Kirkland, WA).
