A buccal gingival sample from either healthy or periodontitis-affected tissue from the premolar/molar maxillary region of each animal was taken using a standard gingivectomy technique, and maintained frozen in RNAlater solution. Total RNA was isolated from each gingival tissue using a standard procedure as we have described and tissue RNA samples submitted to the microarray core to assess RNA quality analyze the transcriptome using the GeneChip® Rhesus Macaque Genome Array (Affymetrix) (Meka, Bakthavatchalu, Sathishkumar, Lopez, Verma, Wallet, Bhattacharyya, Boyce, Handfield, Lamont, Baker, Ebersole and Kesavalu, Gonzalez, Stromberg, Huggins, Gonzalez-Martinez, Novak and Ebersole, 2011 (
link)). Individual samples were used for gene expression analyses.
Table 1 provides an overview of the genes whose expression was evaluated in the gingival tissues as related to M1/2, M1, and M2 phenotype macrophages. These include an array of cytokine/chemokine responses, as well as various cell surface molecules that provide some discrimination among these phenotypes.
Based upon the microarray outcomes we selected 5 genes and performed a qPCR analysis using a standard technique in our laboratory employing a Roche 480 LightCycler (Gonzalez, John Novak, Kirakodu, Stromberg, Shen, Orraca, Gonzalez-Martinez and Ebersole, 2013 (
link)). Primers were prepared for IL6 (forward - CCTGAACCAACCACAAATGC; reverse – GGACTGCAGGAACTCCTTAAA; amplicon 114 bp), CXCL13 (forward – AGTCT GGAAGAAGAACAAGTCAG; reverse – GGAACTGGTGGAGTTGAAGAA; amplicon 108 bp), CCL19 (forward - GCTACCTTCTCATCAAGGATGG; reverse – GTTCTACCCAG GACTGGTCT; amplicon 102 bp), CD14 (forward - GCCCTAAACTCCCTCAATCTG; reverse – CAGTCTGTTGCAGCTGAGAT; amplicon 99 bp) and IL1R2 (forward – TTTCAGACACTACGCACCAC; reverse – ACCGTCTGTGCATCCATATTC; amplicon 118 bp) and GAPDH (forward – GGTGTGAACCATGAGAAGTATGA; reverse – GAGTCCTTCCACGATACCAAAG; amplicon 123 bp) genes, designed using software PrimerQuest at Integrated DNA Technologies website (
www.idtdna.com) and were synthesized by Integrated DNA Technologies, Inc (Coralville, IA). The level of message was determined (Gonzalez, John Novak, Kirakodu, Stromberg, Shen, Orraca, Gonzalez-Martinez and Ebersole, 2013 (
link), Gonzalez, Novak, Kirakodu, Orraca, Chen, Stromberg, Gonzalez-Martinez and Ebersole, 2014 (
link), Ebersole, Kirakodu, Novak, Stromberg, Shen, Orraca, Gonzalez-Martinez, Burgos and Gonzalez, 2014 ) and those levels compared across the RNA samples prepared from each of the healthy groups and the 2 periodontitis groups.
Gonzalez O.A., Novak M.J., Kirakodu S., Stromberg A., Nagarajan R., Huang C.B., Chen K.C., Orraca L., Martinez-Gonzalez J, & Ebersole J.L. (2015). Differential Gene Expression Profiles Reflecting Macrophage Polarization in Aging and Periodontitis Gingival Tissues. Immunological investigations, 44(7), 643-664.