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Chemokine receptor mRNA expression levels were investigated by quantitative real-time polymerase chain reaction (qRT-PCR). RNA was isolated from fresh frozen tissue of ACCs (n = 9), normal adrenal glands (n = 4), aldosterone-producing adenomas (n = 11), cortisol-producing adenomas (n = 10), and non-functioning adenomas (n = 3), using the RNeasy Lipid Tissue Minikit (Qiagen, Hilden, Germany). Of the 33 tumor samples, 12 corresponded to samples investigated also by qRT-PCR (see below). Reverse transcription of RNA was performed using the QuantiTect Reverse Transcription Kit (Qiagen), as previously described [31 (link)]. We used the Taqman Gene Expression assays for CCR7 (Hs01013469_m1) from Applied Biosystems (Darmstadt, Germany). Endogenously expressed β-actin (Hs9999903_m1) was used for normalization. A quantity of 40 ng cDNA was used for each PCR reaction. Transcript levels were determined using the TaqMan Gene Expression Master Mix (Applied Biosystems), the CFX96 real-time thermocycler (Bio-rad, Hercules, CA, USA) and Bio-Rad CFX Manager 2.0 software. Cycling conditions were 95 °C for 3 min followed by 50 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. Using the ΔCT method, the gene expression levels were normalized to those of β-actin, as previously described [32 (link)].
Analysis of publicly available TCGA and GTEx data was performed using GEPIA2 [33 (link),34 (link),35 (link)]. For mRNA expression, analysis was based on “TCGA tumors versus (TCGA normal + GTEx normal)” and expression data was log2(TPM+1)-transformed. Pearson’s correlation of publicly available mRNA expression datasets was performed.

In our cohort, the mRNA expression of RRM1 and RRM2 were evaluated by quantitative real-time PCR (qRT-PCR). In brief, RNA was isolated from fresh-frozen tissue samples using the RNeasy Lipid Tissue Minikit (Qiagen, Hilden, Germany). Reverse transcription of 1 μg of RNA was performed using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s recommendations. Predesigned Taqman^® gene expression assays for RRM1 (Hs01040698_m1) and RRM2 (Hs00357247_g1) (Applied Biosystems, Darmstadt, Germany) were used. Beta actin (Hs9999903_m1) expression was used for normalization. Remaining conditions for qRT-PCR were applied as previously published [16 (link)]. Transcript levels were determined using Bio-Rad CFX Manager 2.0 software and normalized to those of the housekeeping gene using the ΔCT method (Pfaffl Method), as previously described [16 (link)].

Chemokine receptor mRNA expression levels were investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Adrenocortical tissue is composed of different cell entities and leukocyte infiltration in tumor tissue might have influenced chemokine receptor levels detected by qRT-PCR. We therefore also analyzed the chemokine receptor profile in a total of 13 adrenocortical NCI-H295 cancer cell line samples obtained from 3 different sources. RNA was isolated from fresh frozen tissue of eighteen ACCs (not included in the IHC cohort) and four normal human adrenal glands using the RNeasy Lipid Tissue Minikit (Qiagen, Hilden, Germany) and from the human adrenocortical cancer cell line NCI-H295 using the RNeasy Mini Kit (Qiuagen). Reverse transcription of RNA was performed using the QuantiTect Reverse Transcription Kit (Qiagen), as previously described (34 (link)). The following Taqman Gene Expression assays from Applied Biosystems (Darmstadt, Germany) were used to analyze the chemokine receptor profile: CCR1 (Hs 00928897_s1), CCR2 (Hs 00704702_s1), CCR3 (Hs 01847760_s1), CCR4 (Hs 00747615_s1), CCR5 (Hs99999149_s1), CCR6 (Hs 10890706_s1), CCR7 (Hs01013469_m1), CCR8 (Hs 00174764_m1), CCR9 (Hs01890924_s1), CCR10 (Hs00706455_s1), CCR11 (Hs00664347_s1), CXCR1 (Hs 01921207_s1), CXCR2 (Hs 01891184_s1), CXCR3 (Hs01847760_s1), CXCR4 (Hs00607978_s1), CXCR5 (Hs00540548_s1), CXCR6 (Hs01890898_s1), CXCR7 (Hs00664172_s1) and CX3CR1 (Hs 01922583_s1). Endogenously expressed β-actin (Hs9999903_m1) was used for normalization. 40 ng cDNA was used for each PCR reaction. qRT-PCR was performed three times for each cell line. Transcript levels were determined using the TaqMan Gene Expression Master Mix (Applied Biosystems), the CFX96 real-time thermocycler (Bio-rad, Hercules, CA, USA) and Bio-Rad CFX Manager 2.0 software. Cycling conditions were 95°C for three min followed by 50 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Using the ΔCT method, the gene expression levels were normalized to those of β-actin, as previously described (35 (link)).