Total RNA was prepared from tonsillar tissue using a reagent (TRIzol; Invitrogen; Carlsbad, CA) following the manufacturer’s instructions. Isolated total RNA was quantified using a spectrophotometer (model DU-530; Beckman; Fullerton, CA). Aliquots of total RNA (1 μg) were reverse-transcribed using random primers and a reverse transcriptase (Superscript II-Reverse Transcriptase; Invitrogen) according to the manufacturer’s protocol. Complementary DNA equivalent to 20 ng total RNA were subjected to real-time polymerase chain reaction (PCR) analysis (MX4000; Stratagene; La Jolla, CA) following the manufacturer’s protocol. PCR primers (Invitrogen) and Taqman probes (Biosearch Technologies; Novato CA) for LT1-R, LT2-R, and β-actin were designed using appropriate software (Beacon Designer, version 2.0; Premier Biosoft International; Palo Alto, CA). The primer and probe for LT1-R were as follows: forward primer, 5′-TTATGTTCAC-AAAGGCATTTGG-3′; reverse primer, 5′- GCTCATGGCTGTCATAAAGAA −3′; and Taqman probe, 5′-FAM- TGGTGACTTCTTGTGCCGCCTC-BHQ-1–3′. The primer and probe for LT2-R were as follows: forward primer, 5′- ACTATATTGCCTTGGTGGTGGG-3′; reverse primer, 5′- ATGATGGTGGTCAGTGCCTTC-3′; and Taqman probe, 5′-(FAM)- TGTGAGAAACCCGCAGCCCCGA-(BHQ-1)-3′. The primer and probe for β-Actin were as follows: forward primer, 5′-GACTACCTCATGAAGATCCTCACC-3′; reverse primer, 5′-TCTCCTTAATGTCAC GCACGATT-3′; and Taqman probe, 5′-FAM-CGGCTACAGCTTCACCACCACGG-BHQ-1–3′. Each reaction (25 μL) contained 2.5 μL reaction buffer (10×), 6 mmol/L MgCl2, 0.2 μmol/L deoxynucleoside triphosphate, 0.6 μmol/L each primer, 0.25 μL Taq DNA polymerase (SureStart; Stratagene), and 2 μL complementary DNA dilutions. The cycling condition consisted of 1 cycle at 95°C for 10 min and 40 two-segment cycles (ie, 95°C for 30 s and 55°C for 60 s). Standard curves for the target gene (ie, LT1-R or LT2-R) and the housekeeping gene (ie, β-actin) were performed for each assay. Briefly, 10-fold serial dilutions of control complementary DNA were amplified (MX-4000 PCR machine; Stratagene). The cycle time (ie, the initial amplification cycle) of each standard dilution was plotted against standard complementary DNA copy numbers. Based on the standard curves for each gene, the sample complementary DNA copy number was calculated according to the sample cycle time value. Finally, each of the calculated copy numbers for either LT1-R or LT2-R was normalized against the corresponding β-actin copy numbers. Standard curves and PCR results were analyzed using the software of the PCR machine (MX4000; Stratagene).