PARENT SESSION
Gene Regulation & Function

ASSESSMENT OF cDNA QUALITY AFTER LASER CAPTURE MICRODISSECTION OF RHESUS MONKEY ENDOMETRIUM.

Okulicz, William^1,2, Torres, Mira³, Ace, Christopher¹, ^{1 2 3}

ABSTRACT- Laser capture microdissection (LCM) allows studies on cell-type and regional differences in gene expression within complex tissues. Although RNA can be isolated from such tissue, a means of assessing the quality of the material can be problematic, particularly when there are tissue limitations. The objective of our study was to identity an approach that would allow such an assessment of quality prior to further gene expression analysis. Artificial menstrual cycles were created in rhesus monkeys and endometria were collected during an adequate mid-secretory phase (day 21-23). LCM was used to harvest specific cell-types (glandular epithelia or stroma) from either the functionalis or basalis region of the endometrium. Following reverse transcription and second strand synthesis, we ligated primer-specific adaptors to the cDNA population. One round of amplification resulted in approximately a 75-fold enrichment. Although prior to amplification polymerase chain reaction (PCR) analysis for 18S RNA could not be detected in some samples, a clear distinct band was observed following amplification. Thus, quantity rather than quality resulted in a false negative. In addition, a cDNA smear was only observed following amplification and was used to estimate the relative size range of a given population. The 18S RNA fragment size of 324bp could be easily detected in a cDNA population whose average size was either 350bp or 650bp. The G3PDH fragment size of 983bp could, however, only be detected in the cDNA population with the larger average size range. Our results indicate that a single amplification coupled with PCR detection of an appropriate housekeeping gene can provide an important guide to the quality of the cDNA population prepared from tissue harvested by LCM. In addition, this single amplification step provides considerable material for subsequent gene expression studies. (Supported by a grant from the NICHD (HD31620, WCO)

KEY WORDS: microdissection, endometrium, monkey, cDNA