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

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.

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

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0241821
Author(s):  
Yi-Hui Gu ◽  
Xi-Wei Cui ◽  
Jie-Yi Ren ◽  
Man-Mei Long ◽  
Wei Wang ◽  
...  
Keyword(s):  
Schwann Cell ◽  
Cell Lines ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Neurofibromatosis Type ◽  
Internal Reference ◽  
Real Time Quantitative Pcr ◽  
Internal Reference Gene

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.

scholarly journals IFT80 Improves Invasion Ability in Gastric Cancer Cell Line via ift80/p75NGFR/MMP9 Signaling

2018 ◽  
Vol 19 (11) ◽  
pp. 3616 ◽  
Author(s):  
Rui Wang ◽  
Xiaoyan Deng ◽  
Chengfu Yuan ◽  
Hongmei Xin ◽  
Geli Liu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Reverse Transcription ◽  
Gastric Cancer Cell ◽  
Gastric Cancer Cells ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr ◽  
Gastric Cancer Cell Lines

The assembly and maintenance of cilia depend on intraflagellar transport (IFT) proteins, which play an important role in development and homeostasis. IFT80 is a newly defined IFT protein and partial mutation of IFT80 in humans causes diseases such as Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III, both characterized by abnormal skeletal development. However, the role and mechanism of IFT80 in the invasion of gastric cancer is unknown. We established SGC-7901 and MKN-45 gastric cancer cell lines that stably overexpressed IFT80, as verified by quantitative reverse transcription-PCR, Western blot, and immunofluorescence. Matrix metalloproteinase-9 (MMP9) plays an important role in tumor invasion, and its expression was assessed by quantitative reverse transcription-PCR, Western blotting, and immunofluorescence. The invasion ability of IFT80 on SGC-7901 and MKN-45 cells was examined by the Matrigel invasion assay. The relationship between p75NGFR, and the p75NGFR antagonists, PD90780 and IFT80, were detected by quantitative reverse transcription-PCR and Western blotting. We first detected an IFT80 expression pattern, and found that IFT80 was highly expressed in gastric cancer clinical samples. Overexpression of IFT80 in the gastric cancer cell lines, SGC-7901 and MKN-45, led to lengthening cilia. Additionally, overexpression of IFT80 significantly improved proliferation and invasion, but inhibited apoptosis, in gastric cancer cells. We further found that overexpression of IFT80 increased p75NGFR and MMP9 mRNA and protein expression. Treatment with the p75NGFR antagonist PD90780 inhibited the increased invasion ability resulting from overexpression of IFT80 in SGC-7901 and MKN-45 gastric cancer cells. Thus, these results suggest that IFT80 plays an important role in invasion of gastric cancer through regulating the ift80/p75NGFR/MMP9 signal pathways.

High-Throughput Real-Time Quantitative Reverse Transcription PCR

2006 ◽  
Vol 73 (1) ◽  
pp. 15.8.1-15.8.28 ◽  
Author(s):  
Angie L. Bookout ◽  
Carolyn L. Cummins ◽  
David J. Mangelsdorf ◽  
Jean M. Pesola ◽  
Martha F. Kramer
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Reverse Transcription ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr

An Eco-friendly Fabrication of Silver Chloride Nanoparticles (AgCLNPs) using Onopordum acanthium L extract Induces Apoptosis in Breast Cancer MDA-MB-232 Cells

2021 ◽  
Author(s):  
Seyedeh Negin Shahcheraghi ◽  
Seyed Ataollah Sadat Shandiz ◽  
Bahareh Pakpour
Keyword(s):  
Breast Cancer ◽  
Real Time ◽  
Cell Lines ◽  
Reverse Transcription ◽  
Hek293 Cell ◽  
Silver Chloride ◽  
Hek293 Cells ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Onopordum Acanthium

Abstract In the current experimental work, silver chloride nanoparticles (AgClNPs) were fabricated using Onopordum acanthium L extract and their apoptotic and cytotoxicity properties on breast cancer MDA_MB232 and normal HEK293 cell lines were also evaluated. AgClNPs formation was determined by X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) profile. Effect of fabricated AgClNPs on MDA_MB232 and HEK293 cells viability was performed using colorimetric MTT assay. Alterations in the mRNA expression levels of CAD and Bax genes in MDA-MB-232 cells were done using quantitative real-time reverse transcription-PCR (qRT-PCR) method. Subsequently, apoptotic properties were determined using flow cytometry and fluorescence microscopy studies. MTT results investigated that AgCLNPs have a significant dose-dependent lethal activity on MDA_MB232 compared to HEK293 cell lines. Quantitative real-time reverse transcription-PCR (qRT-PCR) results have also shown that AgCLNPs could up-regulate the apoptotic Bax and CAD gene expressions in the MDA_MB232 cells. Additionally, apoptotic assessment was performed by cell cycle analysis, annexin V/PI test, Hoescht 33258 dye, acridine orange and ethidium bromide (AO/EB) staining along with the detection of the reactive oxygen species (ROS) generation. Our results suggest that novel silver chloride nanoparticles fabricated by Onopordum acanthium L extract can display some promising cytotoxic properties through inducing apoptosis pathway.

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