Fig 1.
Overview of the study.
Table 1.
Patient and tumor characteristics.
Table 2.
Nine candidate reference genes and three hypoxia-induced genes evaluated in this study.
Fig 2.
Pre-evaluation of 9 candidate reference genes by RT-qPCR in 10 patients.
(A) Gel electrophoresis of the PCR products for nine candidate reference genes in one patient. Lower (25 bp) and upper (1500 bp) markers are shown in each lane. Gene symbols are indicated. The figure is a composite image where CHCHD1 is from a separate image and the ladder from each image is shown. Vertical lines indicate cropping of the image or different images. (B) Box plots of the arithmetic means of duplicate Cq-values for eight candidate reference genes in 10 patients. Boxes indicate the interquartile range (IQR) with median as the black center bar. Extended vertical bars represents 1.5 x IQR below the first quartile and 1.5 x IQR above the third quartile, and circles mark suspected outliers. (C) geNorm analysis of eight candidate reference genes. Average expression stability (M) of the remaining candidates after stepwise removal of the least stable gene is shown. The least stable gene in each step is indicated below. (D) Stability value of each of the eight candidate reference genes from the NormFinder analysis, where a low value indicates more stable expression.
Fig 3.
Evaluation of overall stability of 5 candidate reference genes by RT-qPCR in 74 patients.
(A) Box plots of the arithmetic means of duplicate Cq-values for five candidate reference genes in 74 patients. Boxes indicate the interquartile range (IQR) with median as the black center bar. Extended vertical bars represent 1.5 x IQR below the first quartile and 1.5 x IQR above the third quartile, and circles mark suspected outliers. (B) geNorm analysis of five candidate reference genes. Average expression stability (M) of the remaining candidates after stepwise removal of the least stable gene is shown. The least stable gene in each step is indicated below. (C) Stability value of each of the five candidate reference genes from the NormFinder analysis, where a low value indicates more stable expression. (D) geNorm pairwise variation (V) analysis to determine the sufficient number of reference genes in the normalization factor. Pairwise variation for two sequential normalization factors (Vn/n+1) from the two most stable genes to all five genes. The horizontal line indicates the cut-off value (V = 0.15), for which inclusion of more genes has no significant effect on the normalization factor.
Fig 4.
Evaluation of stability across subgroups for 5 candidate reference genes by RT-qPCR in 74 patients.
NormFinder analyses of the stability of five candidate reference genes across patient subgroups. The subgroups assessed were: low (n = 49) and high (n = 25) tumor stage (FIGO 1B-2B vs. 3A-4A), with (n = 32) and without (n = 42) lymph node (LN) involvement at diagnosis, below (n = 36) and above (n = 36) a median tumor volume of 44.6 cm3, with (n = 32) or without (n = 42) treatment recurrence at five years, and different hypoxia status represented by below (n = 16) and above (n = 16) a median ABrix.
Fig 5.
CHCHD1, SRSF9 and TMBIM6 as reference genes in studies of hypoxia-induced gene expression in cervical cancer patients.
(A) Gel electrophoresis of the PCR products for the three hypoxia-induced genes DDIT3, ERO1A, and STC2. Lower (25 bp) and upper (1500 bp) markers are shown in each lane. The figure is derived from one image, and vertical lines indicate cropping of the image. Cumulative incidence of disease progression for 74 patients divided into low (< 67% percentile) and high (≥ 67% percentile) STC2 expression based on (B) Illumina expression data and (C) RT-qPCR data normalized with CHCHD1, SRSF9 and TMBIM6 (-ΔCq). 60 months recurrence probability, P-values from Gray’s test and number of patients at risk are indicated. Death from other causes than cervical cancer was included as a competing event (n = 5). (D) Intra-tumor variability in STC2 expression levels measured by RT-qPCR across eight independent tumors with 2–4 biopsies per tumor, i.e. in total 22 biopsies. Measurement of STC2 was unsuccessful for one of the biopsies for tumor 4. STC2 data were normalized with CHCHD1, SRSF9 and TMBIM6. The samples were classified into a high and low expression group using the same cut-off as in Fig 5C (i.e. –ΔCq = -4.46). Different biopsies from the same tumor have been plotted with the same color to ease the interpretation of the figure.
Table 3.
Association between gene expression and tumor hypoxia status (ABrix).