Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue

Smith, T.; Ahmed, AbdelKarem Omneya I; Irlam, Joely J.; Lane, Brian R.; Valentine, Helen; Bibby, Becky A.S.; Denley, Helen; Choudhury, Ananya; West, Catharine M L

doi:10.1038/s41598-020-74380-7

Cited by 12 publications

(11 citation statements)

References 30 publications

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“…Normal and tumour tissues from such patients were analysed by qRT-PCR for the following 12 genes; six from RNA-seq: CLDN1 , MAGEB2 , CD24 , CEACAM6 , IL1B and ISG15 and six from RNA-seq and proteomics cross-linking: AKR1C3 , TNFAIP2 , RAB7A , LGALS3BP , PSCA and SSRP1 . The endogenous genes TBP and IPO8 were included, as described [ 41 ] ( Figure 3 A). The only gene able to distinguish the resistant versus sensitive group was MAGEB2 ( Figure 3 B,C).…”

Section: Resultsmentioning

confidence: 99%

Transcriptomic and Proteomic Profiles for Elucidating Cisplatin Resistance in Head-and-Neck Squamous Cell Carcinoma

García-Mayea

Benítez-Álvarez

Sánchez-García

et al. 2022

Cancers

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To identify the novel genes involved in chemoresistance in head and neck squamous cell carcinoma (HNSCC), we explored the expression profiles of the following cisplatin (CDDP) resistant (R) versus parental (sensitive) cell lines by RNA-sequencing (RNA-seq): JHU029, HTB-43 and CCL-138. Using the parental condition as a control, 30 upregulated and 85 downregulated genes were identified for JHU029-R cells; 263 upregulated and 392 downregulated genes for HTB-43-R cells, and 154 upregulated and 68 downregulated genes for CCL-138-R cells. Moreover, we crossed-checked the RNA-seq results with the proteomic profiles of HTB-43-R (versus HTB-43) and CCL-138-R (versus CCL-138) cell lines. For the HTB-43-R cells, 21 upregulated and 72 downregulated targets overlapped between the proteomic and transcriptomic data; whereas in CCL-138-R cells, four upregulated and three downregulated targets matched. Following an extensive literature search, six genes from the RNA-seq (CLDN1, MAGEB2, CD24, CEACAM6, IL1B and ISG15) and six genes from the RNA-seq and proteomics crossover (AKR1C3, TNFAIP2, RAB7A, LGALS3BP, PSCA and SSRP1) were selected to be studied by qRT-PCR in 11 HNSCC patients: six resistant and five sensitive to conventional therapy. Interestingly, the high MAGEB2 expression was associated with resistant tumours and is revealed as a novel target to sensitise resistant cells to therapy in HNSCC patients.

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Section: Resultsmentioning

confidence: 99%

Transcriptomic and Proteomic Profiles for Elucidating Cisplatin Resistance in Head-and-Neck Squamous Cell Carcinoma

García-Mayea

Benítez-Álvarez

Sánchez-García

et al. 2022

Cancers

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“…Several publications described the identification of reference genes for bladder cancer [ 53 , 54 , 55 ]. For example, using 9 candidate genes and 14 tumor and control tissues, Ohl et al found SDH and TBP as most stable genes using qPCR ( Table S3 ) [ 53 ].…”

Section: Discussionmentioning

confidence: 99%

Improved Bladder Tumor RNA Isolation from Archived Tissues Using Methylene Blue for Normalization, Multiplex RNA Hybridization, Sequencing and Subtyping

Köhler

Brandl

Strissel

et al. 2022

IJMS

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Methylene blue (MB) is a dye used for histology with clinical importance and intercalates into nucleic acids. After MB staining of formalin fixed paraffin embedded (FFPE) muscle invasive bladder cancer (MIBC) and normal urothelium, specific regions could be microdissected. It is not known if MB influences RNA used for gene expression studies. Therefore, we analyzed MIBC using five different RNA isolation methods comparing patient matched FFPE and fresh frozen (FF) tissues pre-stained with or without MB. We demonstrate a positive impact of MB on RNA integrity with FF tissues using real time PCR with no interference of its chemical properties. FFPE tissues showed no improvement of RNA integrity, which we propose is due to formalin induced nucleotide crosslinks. Using direct multiplex RNA hybridization the best genes for normalization of MIBC and control tissues were identified from 34 reference genes. In addition, 5SrRNA and 5.8SrRNA were distinctive reference genes detecting <200 bp fragments important for mRNA analyses. Using these normalized RNAs from MB stained MIBC and applying multiplex RNA hybridization and mRNA sequencing, a minimal gene expression panel precisely identified luminal and basal MIBC tumor subtypes, important for diagnosis, prognosis and chemotherapy response.

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“…Each of them has its mathematical strategy and thus has its pros and cons (Robledo et al, 2014). As an example, geNorm is considered powerful for small sample sizes while NormFinder is recommended for samples with large numbers (Smith et al, 2020). Analyzing the stability of five candidate reference genes in exosomes derived from liver and breast cancer cell lines showed that the stability ranking provided by geNorm and BestKeeper are fairly constant similar to the ranking obtained by Delta Ct and NormFinder.…”

Section: Discussionmentioning

confidence: 99%

Validation of common reference genes stability in exosomal mRNA‐isolated from liver and breast cancer cell lines

Gorji-Bahri

Moradtabrizi

Vakhshiteh

et al. 2021

Cell Biology International

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Accurate relative gene expression analysis by reverse transcription‐quantitative polymerase chain reaction relies on the usage of suitable reference genes for data normalization. The RNA content of small extracellular vesicles including exosomes is growingly considered as cancer biomarkers. So, reliable relative quantification of exosomal messenger RNA (mRNA) is essential for cancer diagnosis and prognosis applications. However, suitable reference genes for accurate normalization of a target gene in exosomes derived from cancer cells are not depicted yet. Here, we analyzed the expression and stability of eight well‐known reference genes namely GAPDH, B2M, HPRT1, ACTB, YWHAZ, UBC, RNA18S, and TBP in exosomes‐isolated from the liver (Huh7, HepG2, PLC/PRF/5) and breast (SK‐BR‐3) cancer cell lines using five different algorithms including geNorm, BestKeeper, Delta Ct, NormFinder, and RefFinder. Our results showed that ACTB, TBP, and HPRT1 were not expressed in exosomes‐isolated from studied liver and breast cancer cell lines. The geNorm and BestKeeper algorithms indicated GAPDH and UBC as the most stable candidates. Moreover, Delta Ct and NormFinder algorithms showed YWHAZ as the most stable reference genes. Comprehensive ranking calculated by the RefFinder algorithm also pointed out GAPDH, YWHAZ, and UBC as the first three stable reference genes. Taken together, this study validated the common reference genes stability in exosomal mRNA derived from liver and breast cancer cell lines for the first time. We believe that this study would be the first step in finding more stable reference genes in exosomes that triggers more accurate detection of exosomal biomarkers.

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Selection of endogenous control genes for normalising gene expression data derived from formalin-fixed paraffin-embedded tumour tissue

Cited by 12 publications

References 30 publications

Transcriptomic and Proteomic Profiles for Elucidating Cisplatin Resistance in Head-and-Neck Squamous Cell Carcinoma

Transcriptomic and Proteomic Profiles for Elucidating Cisplatin Resistance in Head-and-Neck Squamous Cell Carcinoma

Improved Bladder Tumor RNA Isolation from Archived Tissues Using Methylene Blue for Normalization, Multiplex RNA Hybridization, Sequencing and Subtyping

Validation of common reference genes stability in exosomal mRNA‐isolated from liver and breast cancer cell lines

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