The procedure to measure mRNA levels involved a comparison of values obtained from experimental samples (brain extracts) to those obtained for a set of calibration standards. The calibration standards had known amounts of an in vitro sense transcript whose concentration was determined by optical absorbance at 260 nm. The set of calibration standards included those with no added sense transcript and those that contained between 1.25 and 80 pg of the sense transcript [21]. To determine the total attomoles of each mRNA in each extract, the amounts calculated from the standard curves were multiplied by 2.7 for POMC, 1.25 for ppDyn, 3.5 for KOR, 4.3 for orexin, 5.0 for AVP, 2.4 for AVP V1b receptor, and 1.2 for dopamine D2 receptor to correct for the difference in length between the sense transcript and full-length mRNA. A new standard curve was generated each time experimental samples were analyzed and all extracts of a particular tissue were assayed for each mRNA as a group in a single assay.
