Skip to main content
. Author manuscript; available in PMC: 2013 May 15.
Published in final edited form as: J Neurosci Methods. 2012 Mar 10;206(2):200–204. doi: 10.1016/j.jneumeth.2012.02.023

Fig. 1. Pre-amplification of cDNA from Cell Lysate Buffer®proportionally enhances LCM/qrt-PCR of BMEC.

Fig. 1

At day 16 following MOG35–55 peptide immunization to induce EAE (disease score ~ 2.0), mouse cerebellar tissue was processed for immuno-LCM, and BMEC captured by 1000 LCM shots then extracted with Cell Lysate Buffer®. BMEC extract then underwent reverse transcription reaction without prior RNA isolation, and equal aliquots of the resulting cDNA were either not pre-amplified or subject to 14 cycles of pre-amplification prior to single-plex qrt-PCR detection with TaqMan® probes/primers for the genes GAPDH, VCAM1 and CCL2. Each qrt-PCR reaction was run in duplicate (technical replicates), and the mean Ct values for each gene are depicted. Pre-amplification of 14 cycles with extract from 1000 shots of LCM-derived BMEC yielded the following mean ΔCt [cDNA – pre-AMP] values: GAPDH (12.087), VCAM1 (12.34), and CCL2 (10.21). Inherent lower expression of CCL2 may explain the lesser-detected amplification of this gene.