Eight genes (B2M, ATP5B, CYC1, GAPDH, SDHA, TOP1, EIF4A2, UBE2D2) were selected with an average Cq of 22.4 (range 20.57 – 24.2) eliminating the ones with lower expression levels. We analyzed these 8 genes by RT-qPCR with c-DNA obtained from 44 OPSCC and 4 normal tonsil/uvula tissue samples. Our study was in compliance with the recent MIQE guidelines for optimum sharing of RT-qPCR data214. A flowchart describing the overall methodology is given in Figure 3.1. Raw Cq values of selected 8 genes were plotted to see an overall variation in expression values (Figure 3.2). We then compared the expression levels of 8 reference genes through BestKeeper, Normfinder, GeNorm/qbase+ and compared the ranking of genes between all the five tools.
The first analysis was conducted with excel-based analysis tool- BestKeeper. The Cq values were referred to as, crossing points (CP) and the overall trend of raw CP values is as given in Figure 3.3. The inter-gene relationships of all the reference genes/housekeeping genes (HKG) were analyzed by repeated pair-wise correlation and their respective coefficients (r) and p values were determined (Table 3.1). Figure 3.4 (A & B) gives geometric means and standard deviation (SD) of each gene across 48 samples. Respective values of SD were plotted to represent the stability of all HKG’s, where stability increases from left to right, in Figure 3.3 (C). A standard deviation of more than 1 makes a gene unsuitable as a reference gene for analysis, which in our data was applicable for GAPDH.
Figure 3.1: Flowchart of reference gene selection
commonly used reference genes were assessed
candidate reference genes with a closer range of raw Cq values were analyzed for their stability through three software
qbase+. Final candidates were selected as per geNorm
Figure 3.1: Flowchart of reference gene selection. Expression levels of 11
commonly used reference genes were assessed through NHKC c-DNA. Eight candidate reference genes with a closer range of raw Cq values were analyzed for their stability through three software- Normfinder, BestKeeper and geNorm qbase+. Final candidates were selected as per geNorm-qbase+ analysis.
Expression levels of 11 DNA. Eight candidate reference genes with a closer range of raw Cq values were analyzed
Figure 3.2: Reference gene expression of 8 genes in samples of the oral cavity and oropharynx. Raw Cq values are represented as box-whisker plot based on
individual expression values in 44 tumor and 4 control samples. The range of Cq values differed from more than 7 (GAPDH) to less than 4 (TOP1) cycle numbers.
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Table 3.1: Descriptive statistics of pair-wise correlation (Pearson’s correlation, r)
GAPDH ATP5B EIF4A2 TOP1 SDHA CYC1 UBE2D2 B2M vs. HKG 1 HKG 2 HKG 3 HKG 4 HKG 5 HKG 6 HKG 7 HKG 8 HKG 2 0.849 - - - - p- value 0.001 - - - - HKG 3 0.633 0.785 - - - - p- value 0.001 0.001 - - - - HKG 4 0.848 0.911 0.799 - - - - - p- value 0.001 0.001 0.001 - - - - - HKG 5 0.783 0.883 0.841 0.876 - - - - p- value 0.001 2.14E- 16 0.001 0.001 - - - - HKG 6 0.844 0.92 0.751 0.861 0.882 - - - p- value 0.001 0.001 0.001 0.001 0.001 - - - HKG 7 0.592 0.776 0.681 0.751 0.76 0.695 - - p- value 0.001 0.001 0.001 0.001 0.001 0.001 - - HKG 8 0.375 0.533 0.489 0.506 0.526 0.556 0.66 - p- value 0.009487 0.001 0.001 0.001 0.001 0.001 0.001 -
Bestkeeper correlates each gene to the other by pearson’s pairwise correlation analysis and the respective pearson’s coefficient (r) along with its p value is given for each comparison. (HKG-Housekeeping Gene)
Figure 3.3: BestKeeper Analysis- CP of Housekeeping Genes
Raw threshold cycle numbers are represented as crossing points (CP) in BestKeeper analysis and the graph show the overall distribution of CP per sample per gene across the data set. The respective genes are represented with color coded signs given on the right.
Figure 3.4: BestKeeper Analysis- Housekeeping Gene stability
A) Geometric means of all CP values per gene are given with their respective standard errors. B) Overall standard deviation of gene
Normfinder algorithm227, an excel add-in, returns a stability value for each reference gene and thus ranks them based on their variation of expression across a given sample set. Figure 3.5 (A) depicts the inter-group variation of each gene, whereby the samples were grouped as HPV-active, -inactive, -negative and controls. The intra-group variations are as depicted by the error bars of each gene. The most stably expressed gene was CYC1 with a stability value of 0.140 and top two candidate reference genes with a cumulative stability value of 0.111 were, CYC1 and SDHA. The stability values of all genes are as given in Figure 3.5 (B) with the two best candidate genes highlighted in a square. The stability increases from left to right in Figure 3.5 (B).
GeNorm is now available in combination with the RT-qPCR analysis software qbase+ and we conducted our gene expression analysis on the same platform. One novel feature of GeNorm which was utilized in assessing the expression variations was the introduction of Inter-run calibration. Two samples were consistently used as IRC’s and the calibration factors were calculated as a geometric mean of these two IRC’s per plate per gene (Figure 3.6 (A)). The closer the values of calibration factors per gene, the less are the inter-run variations. GeNorm also ranked the given set of genes based on their stability values (M value) which is given in Figure 3.6 (B). The Stability Values of Reference genes ranged from 0.8327 – 1.8016, which is higher than the set threshold of 0.75. Figure 3.6 (C) depicts the stability values of different number of reference genes when considered together. This geNorm V values suggested that optimum number of reference genes for this analysis are 3 with top candidate being- ATP5B, SDHA and TOP1.
Figure 3.5: Normfinder Analysis
A) The intergroup variation of 8 reference genes across 4 major groups: HPV
Controls. The error bars indicate intra
Figure 3.5: Normfinder Analysis
A) The intergroup variation of 8 reference genes across 4 major groups: HPV-active, HPV-inactive, HPV-negative and Controls. The error bars indicate intra-group variation.
A) The intergroup variation of 8 reference genes across 4 negative and
Figure 3.6: GeNorm Analysis
A) The stability values (M), coefficient of variation across the sample set and the inert-run calibration factors per plate are given for each gene, respectively. The values are as calculated by the qbase+ software.
B) Increasing order of stability of ref respective M values.
C) GeNorm returned V values for determining the optimum number of reference genes. The horizontal grey line indicates the threshold cutoff for maximum stability of reference genes. GeNorm sugges
ATP5B, TOP1 and SDHA as the optimum normalization factor.
Figure 3.6: GeNorm Analysis
A) The stability values (M), coefficient of variation across the sample set and the run calibration factors per plate are given for each gene, respectively. The values are as calculated by the qbase+ software.
B) Increasing order of stability of reference genes from left to right based on C) GeNorm returned V values for determining the optimum number of reference genes. The horizontal grey line indicates the threshold cutoff for maximum stability of reference genes. GeNorm suggested the use of geometric means of 3 reference genes ATP5B, TOP1 and SDHA as the optimum normalization factor.
C
A) The stability values (M), coefficient of variation across the sample set and the run calibration factors per plate are given for each gene, respectively. The values erence genes from left to right based on C) GeNorm returned V values for determining the optimum number of reference genes. The horizontal grey line indicates the threshold cutoff for maximum stability of ted the use of geometric means of 3 reference genes-
In view of the recently reported refFinder and Delta Ct method for evaluating most stable reference genes, we analyzed our data set through web-based tool refFinder and compared the gene ranking as given in Table 3.2. The RefFinder tool calculates stability values for each analysis tool (BestKeeper, Normfinder, GeNorm and Delta Ct) and returns a comprehensive ranking list of genes. The refFinder’s stability values and the ranking of reference genes for GeNorm and Normfinder tools were inaccurate when compared to our previously calculated values. The Delta Ct stability values are as given by the refFinder. The genes given in bold were final candidates for normalization and depict variation in their rankings.