Reference gene evaluation for normalization of gene expression studies with lymph tissue and node‑derived stromal cells of patients with oral squamous cell carcinoma

Abstract

Profiling studies using reverse transcription quantitative PCR (RT-qPCR) require reliable normalization to reference genes to accurately interpret the results. A stable reference gene panel was established to profile metastatic and non-metastatic lymph nodes in patients with oral squamous cell carcinoma. The stability of 18S ribosomal RNA (18SrRNA), ribosomal Protein Lateral Stalk Subunit P0 (RPLP0), ribosomal Protein L27 (RPL27), TATA-box binding protein (TBP), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), beta-actin (ACTB), glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and vimentin (VIM) was evaluated, as reference genes for profiling patient-derived lymph node stromal cells (LNSCs; N=8; N0:6, N+:2) and lymph node tissues (Patients:14, Nodes=20; N0:7; N+:13). The genes were initially assessed based on their expression levels, specificity, and stability rankings to identify the best combination of reference genes. VIM was excluded from the final analysis because of its low expression (high quantification cycle >32) and multiple peaks in the melting curve. The stability analysis was performed using Reffinder, which utilizes four tools; geNorm, NormFinder, BestKeeper and Comparative ∆Ct methods, thereby enabling the computing of a comprehensive ranking. Evaluation of the gene profiles indicated that while RPLP0 and 18SrRNA were stable in both lymph node tissues and LNSCs, HPRT1, RPL27 were uniquely stable in these tissues whereas ACTB and TBP were most stable in LNSCs. The present study identified the most stable reference gene panel for the RT-qPCR profiling of lymph node tissues and patient-derived LNSCs. The observation that the gene panel differed between the two model systems further emphasized the need to evaluate the reference gene subset based on the disease and cellular context.

Keywords: lymph node stromal cells; lymph nodes; oral squamous cell carcinoma; reference genes.

Figure 1.

Distribution of Cq values in lymph node tissues and LNSCs. (A) A box and whisker plot depicting the Cq distribution in lymph node tissues demonstrated comparatively lower expression for GAPDH, TBP and HPRT1. (B) Higher expression for RPL27, ACTB, 18SrRNA, RPLP0 and VIM. (C) The scatter plot for Cq values for the reference gene with LNSCs revealed varied distribution. (D) 18SrRNA, ACTB, RPLP0 & VIM demonstrated Cq ranging between 17–24. The box represents the interquartile range and the whiskers represent the minimum and maximum Cq values. LNSCs, lymph node stromal cells; TBP, TATA-box binding protein; HPRT1, hypoxanthine phosphoribosyl-transferase 1; RPL27, ribosomal protein L27; ACTB, beta-actin; 18SrRNA, 18S ribosomal RNA; RPLP0, ribosomal protein lateral stalk subunit P0; VIM, vimentin; N+, metastatic; N0, non-metastatic.

Figure 2.

Ranking for reference genes for lymph node tissues. (A-D) The analysis of reference gene stability with four different methods, namely (A) comparative ∆Ct, (B) NormFinder, (C) geNorm and (D) BestKeeper are demonstrated. The stability analysis with comparative ∆Ct, geNorm and NormFinder identified RPLP0 and HPRT1 as the most stable genes except BestKeeper which identified RPL27 and RPLP0 as the most stable. (E) The comprehensive ranking analysis identifies RPLP0, HPRT1, RPL27 and 18SrRNA as the most stable genes, appropriate to be used as reference genes. RPLP0, ribosomal protein lateral stalk subunit P0; HPRT1, hypoxanthine phosphoribosyl-transferase 1; RPL27, ribosomal protein L27; 18SrRNA, 18S ribosomal RNA; ACTB, beta-actin.

Figure 3.

Ranking for reference genes for LNSCs. The analysis was carried out with eight LNSCs developed in-house. (A-E) The comprehensive analysis with (A) comparative ∆Ct, (B) NormFinder, (C) geNorm, and (D) BestKeeper methods ranked (E) 18SrRNA, RPLP0, ACTB and TBP as the most stable genes that can serve as appropriate reference genes for LNSCs. LNSCs, lymph node stromal cells; 18SrRNA, 18S ribosomal RNA; RPLP0, ribosomal protein lateral stalk subunit P0; ACTB, beta-actin; TBP, TATA-box binding protein; HPRT1, hypoxanthine phosphoribosyl-transferase 1; RPL27, ribosomal protein L27; TBP, TATA-box binding protein.