Selection of internal references for RT-qPCR assays in Neurofibromatosis type 1 (NF1) related Schwann cell lines

Real-time quantitative PCR (RT-qPCR) has been widely applied in uncovering disease mechanisms and screening potential biomarkers. Internal reference gene selection determines the accuracy and reproducibility of data analyses. The aim of this study was to identify the optimal reference genes for the relative quantitative analysis of RT-qPCR in fourteen NF1 related cell lines, including non-tumor, benign and malignant Schwann cell lines. The expression characteristics of eleven candidate reference genes (RPS18, ACTB, B2M, GAPDH, PPIA, HPRT1, TBP, UBC, RPLP0, TFRC and RPL32) were screened and analyzed by four software programs: geNorm, NormFinder, BestKeeper and RefFinder. Results showed that GAPDH, the most frequently used internal reference gene, was significantly unstable between various cell lines. The combinational use of two reference genes (PPIA and TBP) was optimal in malignant Schwann cell lines and the use of single reference genes (PPIA or PRLP0) alone or in combination was optimal in benign Schwann cell lines. These recommended internal reference gene selections may improve the accuracy and reproducibility of RT-qPCR in gene expression analyses of NF1 related tumors.

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Internal Reference Gene Selection under Different Hormone Stresses in Multipurpose Timber Yielding Tree Neolamarckia cadamba

Forests ◽

10.3390/f11091014 ◽

2020 ◽

Vol 11 (9) ◽

pp. 1014

Author(s):

Deng Zhang ◽

Jingjian Li ◽

Buye Li ◽

Chunmei Li ◽

Xiaoyang Chen ◽

...

Keyword(s):

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Housekeeping Genes ◽

Gene Families ◽

Internal Reference ◽

Qpcr Data ◽

Reference Gene Selection ◽

Internal Reference Gene ◽

Cell Wall Extension

Neolamarckia cadamba, a member of the Rubiaceae family, is widely distributed throughout South Asia and South China. In order to acquire reliable and repeatable results, the use of a suitable internal reference gene to normalize the RT-qPCR data is essential. In this study, we reported the validation of housekeeping genes to identify the most suitable internal reference gene(s) for normalization of qPCR data obtained among different tissues (bud, leaf, cambium region) under different hormone stresses. Here, ΔCt, geNorm, NormFinder, and BestKeeper analyses were carried out to analyze the normalization of qPCR data of twenty-one reference gene families (ACT, CAC, CYP, EF1α, eIF, FPS1, FBK, GAPDH, RAN, PEPKR1, PP2A, RPL, RPS, RuBP, SAMDC, TEF, Tub-α, Tub-β, UBCE, UBQ, UPL) including 43 genes. The results showed that FPS1, RPL, and FBK were the most stable reference genes across all of the tested samples. In addition, the expression of NcEXPA8, one gene of interest that plays an important role in regulating cell wall extension, under different phytohormone stresses was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene selection under different phytohormone stresses and a foundation for more accurate and widespread use of RT-qPCR in N. cadamba.

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Selection of internal references for transcriptomics and RT-qPCR assays in Neurofibromatosis type 1 (NF1) related Schwann cell lines

10.1101/2020.10.22.350017 ◽

2020 ◽

Author(s):

Yi-Hui Gu ◽

Xi-Wei Cui ◽

Jie-Yi Ren ◽

Man-Mei Long ◽

Wei Wang ◽

...

Keyword(s):

Schwann Cell ◽

Real Time ◽

Cell Lines ◽

Reverse Transcription ◽

Reference Genes ◽

Neurofibromatosis Type ◽

Internal Reference ◽

Quantitative Reverse Transcription Pcr ◽

Reverse Transcription Pcr ◽

Reference Selection

AbstractTranscriptomics has been widely applied in uncovering disease mechanisms and screening potential biomarkers. Internal reference selection determines the accuracy and reproducibility of data analyses. The aim of this study was to identify the most qualified reference genes for the relative quantitation analysis of transcriptomics and real-time quantitative reverse-transcription PCR in fourteen NF1 related cell lines, including non-tumor, benign and malignant Schwann cell lines. The expression characteristics of eleven candidate reference genes (RPS18, ACTB, B2M, GAPDH, PPIA, HPRT1, TBP, UBC, RPLP0, TFRC and RPL32) were screened and analyzed by four software programs: geNorm, NormFinder, BestKeeper and RefFinder. Results showed that GAPDH, the most used internal reference gene, was significantly unstable. The combinational use of two reference genes (PPIA and TBP) was optimal in malignant Schwann cell lines and the use of single reference genes (PPIA or PRLP0) alone or in combination was optimal in benign Schwann cell lines. Our recommended internal reference genes may improve the accuracy and reproducibility of the results of transcriptomics and real-time quantitative reverse-transcription PCR in further gene expression analyses of NF1 related tumors.

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Validation of internal reference genes for relative quantitation studies of gene expression in human laryngeal cancer

PeerJ ◽

10.7717/peerj.2763 ◽

2016 ◽

Vol 4 ◽

pp. e2763 ◽

Cited By ~ 3

Author(s):

Xiaofeng Wang ◽

Jinting He ◽

Wei Wang ◽

Ming Ren ◽

Sujie Gao ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Laryngeal Cancer ◽

Reference Genes ◽

Target Gene ◽

Cancer Tissue ◽

Internal Reference ◽

Target Gene Expression ◽

Internal Reference Gene ◽

Relative Quantitation

BackgroundThe aim of this study was to determine the expression stabilities of 12 common internal reference genes for the relative quantitation analysis of target gene expression performed by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) in human laryngeal cancer.MethodsHep-2 cells and 14 laryngeal cancer tissue samples were investigated. The expression characteristics of 12 internal reference gene candidates (18S rRNA, GAPDH, ACTB, HPRT1, RPL29, HMBS, PPIA, ALAS1, TBP, PUM1, GUSB, and B2M) were assessed by RT-qPCR. The data were analyzed by three commonly used software programs: geNorm, NormFinder, and BestKeeper.ResultsThe use of the combination of four internal reference genes was more appropriate than the use of a single internal reference gene. The optimal combination was PPIA + GUSB + RPL29 + HPRT1 for both the cell line and tissues; while the most appropriate combination was GUSB + RPL29 + HPRT1 + HMBS for the tissues.ConclusionsOur recommended internal reference genes may improve the accuracy of relative quantitation analysis of target gene expression performed by the RT-qPCR method in further gene expression research on laryngeal tumors.

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c-Kit receptor expression in normal human Schwann cells and Schwann cell lines derived from neurofibromatosis type 1 tumors

Journal of Neuroscience Research ◽

10.1002/jnr.20648 ◽

2005 ◽

Vol 82 (4) ◽

pp. 465-471 ◽

Cited By ~ 14

Author(s):

Ian Dang ◽

Julie K. Nelson ◽

George H. DeVries

Keyword(s):

Schwann Cell ◽

Schwann Cells ◽

Cell Lines ◽

Neurofibromatosis Type 1 ◽

Neurofibromatosis Type ◽

Receptor Expression ◽

Kit Receptor ◽

Normal Human

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Internal Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in Potato Tissues

Phyton ◽

10.32604/phyton.2020.08874 ◽

2020 ◽

Vol 89 (2) ◽

pp. 329-344

Author(s):

Gang Li ◽

Yao Zhou ◽

Yaqi Zhao ◽

Yaxue Liu ◽

Yuwei Ke ◽

...

Keyword(s):

Real Time ◽

Reference Gene ◽

Gene Selection ◽

Rt Pcr ◽

Internal Reference ◽

Reference Gene Selection ◽

Internal Reference Gene ◽

Selection For

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Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

Genes ◽

10.3390/genes12070960 ◽

2021 ◽

Vol 12 (7) ◽

pp. 960

Author(s):

Meagan Archer ◽

Jianping Xu

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Quantitative Polymerase Chain Reaction ◽

Specific Method ◽

Experimental Conditions ◽

Reference Gene Selection ◽

Physiological Processes ◽

The Stability

Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.

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Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

Scientific Reports ◽

10.1038/s41598-021-92780-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Madhab Kumar Sen ◽

Kateřina Hamouzová ◽

Pavlina Košnarová ◽

Amit Roy ◽

Josef Soukup

Keyword(s):

Reference Gene ◽

Herbicide Resistance ◽

Reference Genes ◽

Gene Selection ◽

High Yield ◽

Suitable Reference Gene ◽

Grass Species ◽

Experimental Conditions ◽

Qrt Pcr ◽

Suitable Reference

AbstractBromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

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Assessment of an internal reference gene in Rhodobacter sphaeroides grown under cobalt exposure

Journal of Basic Microbiology ◽

10.1002/jobm.200900340 ◽

2010 ◽

Vol 50 (3) ◽

pp. 302-305 ◽

Cited By ~ 7

Author(s):

Luca Losurdo ◽

Francesca Italiano ◽

Massimo Trotta ◽

Raffaele Gallerani ◽

Ruggiero Ceci Luigi ◽

...

Keyword(s):

Reference Gene ◽

Rhodobacter Sphaeroides ◽

Internal Reference ◽

Internal Reference Gene ◽

Cobalt Exposure

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New Model Systems and the Development of Targeted Therapies for the Treatment of Neurofibromatosis Type 1-Associated Malignant Peripheral Nerve Sheath Tumors

Genes ◽

10.3390/genes11050477 ◽

2020 ◽

Vol 11 (5) ◽

pp. 477 ◽

Cited By ~ 3

Author(s):

Kyle B. Williams ◽

David A. Largaespada

Keyword(s):

Schwann Cell ◽

Peripheral Nerve ◽

Neurofibromatosis Type 1 ◽

Single Agent ◽

Neurofibromatosis Type ◽

Mek Inhibitors ◽

Nerve Sheath Tumors ◽

Peripheral Nerve Sheath Tumors ◽

Nerve Sheath

Neurofibromatosis Type 1 (NF1) is a common genetic disorder and cancer predisposition syndrome (1:3000 births) caused by mutations in the tumor suppressor gene NF1. NF1 encodes neurofibromin, a negative regulator of the Ras signaling pathway. Individuals with NF1 often develop benign tumors of the peripheral nervous system (neurofibromas), originating from the Schwann cell linage, some of which progress further to malignant peripheral nerve sheath tumors (MPNSTs). Treatment options for neurofibromas and MPNSTs are extremely limited, relying largely on surgical resection and cytotoxic chemotherapy. Identification of novel therapeutic targets in both benign neurofibromas and MPNSTs is critical for improved patient outcomes and quality of life. Recent clinical trials conducted in patients with NF1 for the treatment of symptomatic plexiform neurofibromas using inhibitors of the mitogen-activated protein kinase (MEK) have shown very promising results. However, MEK inhibitors do not work in all patients and have significant side effects. In addition, preliminary evidence suggests single agent use of MEK inhibitors for MPNST treatment will fail. Here, we describe the preclinical efforts that led to the identification of MEK inhibitors as promising therapeutics for the treatment of NF1-related neoplasia and possible reasons they lack single agent efficacy in the treatment of MPNSTs. In addition, we describe work to find targets other than MEK for treatment of MPNST. These have come from studies of RAS biochemistry, in vitro drug screening, forward genetic screens for Schwann cell tumors, and synthetic lethal screens in cells with oncogenic RAS gene mutations. Lastly, we discuss new approaches to exploit drug screening and synthetic lethality with NF1 loss of function mutations in human Schwann cells using CRISPR/Cas9 technology.

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