When the difference in body weight gain between ad libitum fed tumor-bearing rats and PBS-injected animals reached 10%, eight rats were subjected to a 33% food restriction until they reached the same body weight as the former.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using 500 ul Trizol following manufacturer’s protocol (Invitrogen, Carlsblad, CA, USA)
Label
Cy3
Label protocol
Total RNA extracts from individual animals were purified (RNeasy MinElute Cleanup kit, Qiagen, Courtaboeuf, France) and treated with DNaseI (Ambion, Austin, TX, USA), after which 2.0 μg of purified RNA were retrotranscribed to double stranded cDNA. A pool of cDNA for each experimental group containing an equal amount of cDNA of each individual was used for in vitro transcription to yield multiple copies of amino-allyl-modified antisense RNA (aRNA) using amino-allyl-labeled UTP (Ambion, Austin, TX, USA). Subsequently, aimine groups of the fluorescent Cy3 or Cy5 dyes (Amersham Biosciences, GE Healthcare, Munich, Germany ) were coupled to the a RNA amino allyl groups following manufacturer’s instructions.
Subcutaneous injection between scapulae with phosphate buffered saline (PBS). Animals had ad libitum access to food for the duration of the experiment.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using 500 ul Trizol following manufacturer’s protocol (Invitrogen, Carlsblad, CA, USA)
Label
Cy5
Label protocol
Total RNA extracts from individual animals were purified (RNeasy MinElute Cleanup kit, Qiagen, Courtaboeuf, France) and treated with DNaseI (Ambion, Austin, TX, USA), after which 2.0 μg of purified RNA were retrotranscribed to double stranded cDNA. A pool of cDNA for each experimental group containing an equal amount of cDNA of each individual was used for in vitro transcription to yield multiple copies of amino-allyl-modified antisense RNA (aRNA) using amino-allyl-labeled UTP (Ambion, Austin, TX, USA). Subsequently, aimine groups of the fluorescent Cy3 or Cy5 dyes (Amersham Biosciences, GE Healthcare, Munich, Germany ) were coupled to the a RNA amino allyl groups following manufacturer’s instructions.
Comparison between hypothalami from freely feeding animals and food-restricted weight-matched animals
Data processing
Agilent Feature-extraction software version A.7.5.1. Spot analysis method using Cookie Cutter, pixel outlier rejection based on IQR, non-uniformity outlier flagging on, population outlier flagging on, no background subtraction, spatial detrend on, normalization feature selection by using rank consistency filter, normalization method linear and LOWESS, multiplicative errors of 0.15 for red and 0.25 for green, additive errors of 20 for both channels.
