Research Article
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Year 2025, Volume: 15 Issue: 2, 387 - 391, 30.06.2025

Burhanettin Yalçınkaya , Zeliha Eskin

https://doi.org/10.33808/clinexphealthsci.1596074

Abstract

References

  • Schmitt AM, Chang HY. Long noncoding RNAs in cancer pathways. Cancer Cell 2016;29(4):452–463. https://doi.org/10.1016/j.ccell.2016.03.010.
  • Roberts MC, Holt KE, Del Fiol G, Baccarelli AA, Allen CG. Precision public health in the era of genomics and big data. Nat Med. 2024;30(7):1865-1873. https://doi.org/10.1038/s41591-024-03098-0.
  • Deniz E, Erman B. Long noncoding RNA (lincRNA), a new paradigm in gene expression control. Funct Integr Genomics 2017;17(2–3):135–143. https://doi.org/10.1007/s10142-016-0524-x.
  • Bure I V, Nemtsova M V, Kuznetsova EB. Histone modifications and non-coding RNAs: Mutual epigenetic regulation and role in pathogenesis. Int J Mol Sci. 2022;23(10): 5801. https://doi.org/10.3390/ijms23105801.
  • Rai A, Bhagchandani T, Tandon R. Transcriptional landscape of long non-coding RNAs (lncRNAs) and its implication in viral diseases. Biochim Biophys Acta - Gene Regul Mech. 2024;1867(2):195023. https://doi.org/https://doi.org/10.1016/j.bbagrm.2024.195023.
  • Yang J, Qi M, Fei X, Wang X, Wang K. Lncrna h19: A novel oncogene in multiple cancers. Int J Biol Sci. 2021;17(12):3188–3208. https://doi.org/10.7150/ijbs.62573.
  • Wang Y, Zeng J, Chen W, Fan J, Hylemon PB, Zhou H. Long noncoding RNA H19: A novel oncogene in liver cancer. Non-Coding RNA 2023;9(2):19. https://doi.org/10.3390/ncrna9020019.
  • Malakar P, Shukla S, Mondal M, Kar RK, Siddiqui JA. The nexus of long noncoding RNAs, splicing factors, alternative splicing and their modulations. RNA Biol. 2024;21(1):1–20. https://doi.org/10.1080/15476286.2023.2286099.
  • Nadhan R, Dhanasekaran DN. Decoding the oncogenic signals from the long non-coding RNAs. Onco. 2021;1(2):176–206. https://doi.org/10.3390/onco1020014.
  • Xin X, Li Q, Fang J, Zhao T. LncRNA HOTAIR: A Potential prognostic factor and therapeutic target in human cancers. Front Oncol 2021;11:1–11. https://doi.org/10.3389/fonc.2021.679244.
  • Yang X, Xie Z, Lei X, Gan R. Long non-coding RNA GAS5 in human cancer (Review). Oncol Lett. 2020;20(3):2587–2594. https://doi.org/10.3892/ol.2020.11809.
  • Yang R, Chen J, Wang L, Deng A. LncRNA BANCR participates in polycystic ovary syndrome by promoting cell apoptosis. Mol Med Rep. 2019;19(3):1581–1586. https://doi.org/10.3892/mmr.2018.9793.
  • Dong Y, Yuan H, Jin G. Identification of long non-coding RNA CCAT1 as an oncogene in nasopharyngeal carcinoma. Oncol Lett. 2018;16(2):2750–2756. https://doi.org/10.3892/ol.2018.8969.
  • Gu C, Zou S, He C, Zhou J, Qu R, Wang Q, Qi J, Zhou M, Yan S, Ye Z. Long non‑coding RNA CCAT1 promotes colorectal cancer cell migration, invasiveness and viability by upregulating VEGF via negative modulation of microRNA‑218. Exp Ther Med. 2020; 19(4):2543–2550. https://doi.org/10.3892/etm.2020.8518.
  • Yang L, Zhang X, Liu X. Long non-coding RNA GAS5 protects against Mycoplasma pneumoniaepneumonia by regulating the microRNA-222-3p/TIMP3 axis. Mol Med Rep. 2021;23(5):380. https://doi.org/10.3892/mmr.2021.12019.
  • Luo R, Li L, Hu YX, Xiao F. LncRNA H19 inhibits high glucose-induced inflammatory responses of human retinal epithelial cells by targeting miR-19b to increase SIRT1 expression. Kaohsiung J Med Sci. 2021;37(2):101–110. https://doi.org/10.1002/kjm2.12302.
  • Zhang L, Song X, Wang X, Xie Y, Wang Z, Xu Y, You X, Liang Z, Cao H. Circulating DNA of HOTAIR in serum is a novel biomarker for breast cancer. Breast Cancer Res Treat. 2015;152(1):199–208. https://doi.org/10.1007/s10549-015-3431-2.
  • Jin C, Shi W, Wang F, Shen X, Qi J, Cong H, Yuan J, Shi L, Zhu B, Luo X, Zhang Y, Ju S. Long non-coding RNA HULC as a novel serum biomarker for diagnosis and prognosis prediction of gastric cancer. Oncotarget. 2016;7(32):51763–51772. https://doi.org/10.18632/oncotarget.10107.
  • Ji Q, Zhang L, Liu X, Zhou L, Wang W, Han Z, Sui H, Tang Y, Wang Y, Liu N, Ren J, Hou F, Li Q. Long non-coding RNA MALAT1 promotes tumour growth and metastasis in colorectal cancer through binding to SFPQ and releasing oncogene PTBP2 from SFPQ/PTBP2 complex. Br J Cancer. 2014;111(4):736–748. https://doi.org/10.1038/bjc.2014.383.
  • Xia Y, He Z, Liu B, Wang P, Chen Y. Downregulation of Meg3 enhances cisplatin resistance of lung cancer cells through activation of the WNT/β-catenin signaling pathway. Mol Med Rep. 2015;12(3):4530–4537. https://doi.org/10.3892/mmr.2015.3897.
  • Hu T, Huang H, Shen H, Chen W, Yang Z. Role of long non‑coding RNA MALAT1 in chronic obstructive pulmonary disease. Exp Ther Med. 2020;20(3):2691–2697. https://doi.org/10.3892/etm.2020.8996.
  • Zheng J, Hu L, Cheng J, Xu J, Zhong Z, Yang Y, Yuan Z. LncRNA PVT1 promotes the angiogenesis of vascular endothelial cell by targeting miR-26b to activate CTGF/ANGPT2. Int J Mol Med. 2018;42(1):489–496. https://doi.org/10.3892/ijmm.2018.3595.
  • Zheng Q, Wu F, Dai WY, Zheng DC, Zheng C, Ye H, Zhou B, Chen JJ, Chen P. Aberrant expression of UCA1 in gastric cancer and its clinical significance. Clin Transl Oncol. 2015;17(8):640–646. https://doi.org/10.1007/s12094-015-1290-2.
  • Rahmi KA, Khotimah H, Rohman MS. Utilization of modified touchdown qPCR to enhance sensitivity and specificity for genes with restricted expression and low optimum temperature primers. Biomed Biotechnol Res J. 2024;8(2): 231-237. https://doi.org/10.4103/bbrj.bbrj_111_24
  • Mamedov TG, Pienaar E, Whitney SE, TerMaat JR, Carvill G, Goliath R, Subramanian A, Viljoen HJ. A fundamental study of the PCR amplification of GC-rich DNA templates. Comput Biol Chem. 2008;32(6):452–457. https://doi.org/10.1016/j.compbiolchem.2008.07.021.
  • Liu H, Ye D, Chen A, Tan D, Zhang W, Jiang W, Wang M, Zhang X. A pilot study of new promising non-coding RNA diagnostic biomarkers for early-stage colorectal cancers. 2019;57(7):1073–1083. https://doi.org/doi:10.1515/cclm-2019-0052.
  • El-Helkan B, Emam M, Mohanad M, Fathy S, Zekri AR, Ahmed OS. Long non-coding RNAs as novel prognostic biomarkers for breast cancer in Egyptian women. Sci Rep. 2022;12(1):19498. https://doi.org/10.1038/s41598-022-23938-8.
  • Motlagh PE, Jamali E, Karimi N, Eslami S, Sharifi G, Ghafouri-Fard S. Integrated bioinformatics approaches and expression assays identified new markers in pituitary adenomas. Pathol - Res Pract. 2024;255:155193. https://doi.org/https://doi.org/10.1016/j.prp.2024.155193.
  • Sun X-H, Yang L-B, Geng X-L, Wang R, Zhang Z-C. Increased expression of lncRNA HULC indicates a poor prognosis and promotes cell metastasis in osteosarcoma. Int J Clin Exp Pathol. 2015;8(3):2994–3000.
  • Tan Q, Zuo J, Qiu S, Yu Y, Zhou H, Li N, Wang H, Liang C, Yu M, Tu J. Identification of circulating long non-coding RNA GAS5 as a potential biomarker for non-small cell lung cancer diagnosisnon-small cell lung cancer, long non-coding RNA, plasma, GAS5, biomarker. Int J Oncol 2017;50(5):1729–1738.

Optimizing qPCR Annealing Temperatures for Cancer-Related lncRNAs

Year 2025, Volume: 15 Issue: 2, 387 - 391, 30.06.2025

Burhanettin Yalçınkaya , Zeliha Eskin

https://doi.org/10.33808/clinexphealthsci.1596074

Abstract

Objective: To determine the optimal annealing temperatures to detect copy number variation and expression levels of specific cancer-associated long non-coding RNAs (lncRNAs), to improve the accuracy of clinical tests in precision medicine.
Methods: Gradient qPCR analysis was performed to identify the optimal annealing temperatures for the detection of lncRNAs. These lncRNAs were H19, CCAT1, HOTAIR, NEAT1, MALAT1, PVT1, GAS5, BANCR, UCA1, HULC, and MEG3.
Results: Optimal qPCR annealing temperatures for the detection of lncRNA expression and copy number variations were determined as follows: 62.4°C for PVT1 and BANCR; 60°C for H19, CCAT1-b, HOTAIR, NEAT1, MALAT1; 58°C for GAS5 and HULC; 56.7°C for CCAT1-a, UCA1; and 56°C for MEG3.
Conclusion: In this study, we determined optimal annealing temperatures for some lncRNAs, which is crucial for the precision and accuracy of qPCR used to identify lncRNA expression and copy number changes. These results confirm the optimization of lncRNA analysis by determining optimal annealing temperatures, which is essential for the precision and accuracy of qPCR.

Keywords

cancer lncRNA qPCR annealing temperature

References

  • Schmitt AM, Chang HY. Long noncoding RNAs in cancer pathways. Cancer Cell 2016;29(4):452–463. https://doi.org/10.1016/j.ccell.2016.03.010.
  • Roberts MC, Holt KE, Del Fiol G, Baccarelli AA, Allen CG. Precision public health in the era of genomics and big data. Nat Med. 2024;30(7):1865-1873. https://doi.org/10.1038/s41591-024-03098-0.
  • Deniz E, Erman B. Long noncoding RNA (lincRNA), a new paradigm in gene expression control. Funct Integr Genomics 2017;17(2–3):135–143. https://doi.org/10.1007/s10142-016-0524-x.
  • Bure I V, Nemtsova M V, Kuznetsova EB. Histone modifications and non-coding RNAs: Mutual epigenetic regulation and role in pathogenesis. Int J Mol Sci. 2022;23(10): 5801. https://doi.org/10.3390/ijms23105801.
  • Rai A, Bhagchandani T, Tandon R. Transcriptional landscape of long non-coding RNAs (lncRNAs) and its implication in viral diseases. Biochim Biophys Acta - Gene Regul Mech. 2024;1867(2):195023. https://doi.org/https://doi.org/10.1016/j.bbagrm.2024.195023.
  • Yang J, Qi M, Fei X, Wang X, Wang K. Lncrna h19: A novel oncogene in multiple cancers. Int J Biol Sci. 2021;17(12):3188–3208. https://doi.org/10.7150/ijbs.62573.
  • Wang Y, Zeng J, Chen W, Fan J, Hylemon PB, Zhou H. Long noncoding RNA H19: A novel oncogene in liver cancer. Non-Coding RNA 2023;9(2):19. https://doi.org/10.3390/ncrna9020019.
  • Malakar P, Shukla S, Mondal M, Kar RK, Siddiqui JA. The nexus of long noncoding RNAs, splicing factors, alternative splicing and their modulations. RNA Biol. 2024;21(1):1–20. https://doi.org/10.1080/15476286.2023.2286099.
  • Nadhan R, Dhanasekaran DN. Decoding the oncogenic signals from the long non-coding RNAs. Onco. 2021;1(2):176–206. https://doi.org/10.3390/onco1020014.
  • Xin X, Li Q, Fang J, Zhao T. LncRNA HOTAIR: A Potential prognostic factor and therapeutic target in human cancers. Front Oncol 2021;11:1–11. https://doi.org/10.3389/fonc.2021.679244.
  • Yang X, Xie Z, Lei X, Gan R. Long non-coding RNA GAS5 in human cancer (Review). Oncol Lett. 2020;20(3):2587–2594. https://doi.org/10.3892/ol.2020.11809.
  • Yang R, Chen J, Wang L, Deng A. LncRNA BANCR participates in polycystic ovary syndrome by promoting cell apoptosis. Mol Med Rep. 2019;19(3):1581–1586. https://doi.org/10.3892/mmr.2018.9793.
  • Dong Y, Yuan H, Jin G. Identification of long non-coding RNA CCAT1 as an oncogene in nasopharyngeal carcinoma. Oncol Lett. 2018;16(2):2750–2756. https://doi.org/10.3892/ol.2018.8969.
  • Gu C, Zou S, He C, Zhou J, Qu R, Wang Q, Qi J, Zhou M, Yan S, Ye Z. Long non‑coding RNA CCAT1 promotes colorectal cancer cell migration, invasiveness and viability by upregulating VEGF via negative modulation of microRNA‑218. Exp Ther Med. 2020; 19(4):2543–2550. https://doi.org/10.3892/etm.2020.8518.
  • Yang L, Zhang X, Liu X. Long non-coding RNA GAS5 protects against Mycoplasma pneumoniaepneumonia by regulating the microRNA-222-3p/TIMP3 axis. Mol Med Rep. 2021;23(5):380. https://doi.org/10.3892/mmr.2021.12019.
  • Luo R, Li L, Hu YX, Xiao F. LncRNA H19 inhibits high glucose-induced inflammatory responses of human retinal epithelial cells by targeting miR-19b to increase SIRT1 expression. Kaohsiung J Med Sci. 2021;37(2):101–110. https://doi.org/10.1002/kjm2.12302.
  • Zhang L, Song X, Wang X, Xie Y, Wang Z, Xu Y, You X, Liang Z, Cao H. Circulating DNA of HOTAIR in serum is a novel biomarker for breast cancer. Breast Cancer Res Treat. 2015;152(1):199–208. https://doi.org/10.1007/s10549-015-3431-2.
  • Jin C, Shi W, Wang F, Shen X, Qi J, Cong H, Yuan J, Shi L, Zhu B, Luo X, Zhang Y, Ju S. Long non-coding RNA HULC as a novel serum biomarker for diagnosis and prognosis prediction of gastric cancer. Oncotarget. 2016;7(32):51763–51772. https://doi.org/10.18632/oncotarget.10107.
  • Ji Q, Zhang L, Liu X, Zhou L, Wang W, Han Z, Sui H, Tang Y, Wang Y, Liu N, Ren J, Hou F, Li Q. Long non-coding RNA MALAT1 promotes tumour growth and metastasis in colorectal cancer through binding to SFPQ and releasing oncogene PTBP2 from SFPQ/PTBP2 complex. Br J Cancer. 2014;111(4):736–748. https://doi.org/10.1038/bjc.2014.383.
  • Xia Y, He Z, Liu B, Wang P, Chen Y. Downregulation of Meg3 enhances cisplatin resistance of lung cancer cells through activation of the WNT/β-catenin signaling pathway. Mol Med Rep. 2015;12(3):4530–4537. https://doi.org/10.3892/mmr.2015.3897.
  • Hu T, Huang H, Shen H, Chen W, Yang Z. Role of long non‑coding RNA MALAT1 in chronic obstructive pulmonary disease. Exp Ther Med. 2020;20(3):2691–2697. https://doi.org/10.3892/etm.2020.8996.
  • Zheng J, Hu L, Cheng J, Xu J, Zhong Z, Yang Y, Yuan Z. LncRNA PVT1 promotes the angiogenesis of vascular endothelial cell by targeting miR-26b to activate CTGF/ANGPT2. Int J Mol Med. 2018;42(1):489–496. https://doi.org/10.3892/ijmm.2018.3595.
  • Zheng Q, Wu F, Dai WY, Zheng DC, Zheng C, Ye H, Zhou B, Chen JJ, Chen P. Aberrant expression of UCA1 in gastric cancer and its clinical significance. Clin Transl Oncol. 2015;17(8):640–646. https://doi.org/10.1007/s12094-015-1290-2.
  • Rahmi KA, Khotimah H, Rohman MS. Utilization of modified touchdown qPCR to enhance sensitivity and specificity for genes with restricted expression and low optimum temperature primers. Biomed Biotechnol Res J. 2024;8(2): 231-237. https://doi.org/10.4103/bbrj.bbrj_111_24
  • Mamedov TG, Pienaar E, Whitney SE, TerMaat JR, Carvill G, Goliath R, Subramanian A, Viljoen HJ. A fundamental study of the PCR amplification of GC-rich DNA templates. Comput Biol Chem. 2008;32(6):452–457. https://doi.org/10.1016/j.compbiolchem.2008.07.021.
  • Liu H, Ye D, Chen A, Tan D, Zhang W, Jiang W, Wang M, Zhang X. A pilot study of new promising non-coding RNA diagnostic biomarkers for early-stage colorectal cancers. 2019;57(7):1073–1083. https://doi.org/doi:10.1515/cclm-2019-0052.
  • El-Helkan B, Emam M, Mohanad M, Fathy S, Zekri AR, Ahmed OS. Long non-coding RNAs as novel prognostic biomarkers for breast cancer in Egyptian women. Sci Rep. 2022;12(1):19498. https://doi.org/10.1038/s41598-022-23938-8.
  • Motlagh PE, Jamali E, Karimi N, Eslami S, Sharifi G, Ghafouri-Fard S. Integrated bioinformatics approaches and expression assays identified new markers in pituitary adenomas. Pathol - Res Pract. 2024;255:155193. https://doi.org/https://doi.org/10.1016/j.prp.2024.155193.
  • Sun X-H, Yang L-B, Geng X-L, Wang R, Zhang Z-C. Increased expression of lncRNA HULC indicates a poor prognosis and promotes cell metastasis in osteosarcoma. Int J Clin Exp Pathol. 2015;8(3):2994–3000.
  • Tan Q, Zuo J, Qiu S, Yu Y, Zhou H, Li N, Wang H, Liang C, Yu M, Tu J. Identification of circulating long non-coding RNA GAS5 as a potential biomarker for non-small cell lung cancer diagnosisnon-small cell lung cancer, long non-coding RNA, plasma, GAS5, biomarker. Int J Oncol 2017;50(5):1729–1738.
There are 30 citations in total.

Details

Primary Language English
Subjects Cancer Genetics
Journal Section Articles
Authors

Burhanettin Yalçınkaya 0000-0002-3744-6634

Zeliha Eskin 0009-0007-5142-665X

Early Pub Date June 27, 2025
Publication Date June 30, 2025
Submission Date December 4, 2024
Acceptance Date May 29, 2025
Published in Issue Year 2025 Volume: 15 Issue: 2

Cite

APA Yalçınkaya, B., & Eskin, Z. (2025). Optimizing qPCR Annealing Temperatures for Cancer-Related lncRNAs. Clinical and Experimental Health Sciences, 15(2), 387-391. https://doi.org/10.33808/clinexphealthsci.1596074
AMA Yalçınkaya B, Eskin Z. Optimizing qPCR Annealing Temperatures for Cancer-Related lncRNAs. Clinical and Experimental Health Sciences. June 2025;15(2):387-391. doi:10.33808/clinexphealthsci.1596074
Chicago Yalçınkaya, Burhanettin, and Zeliha Eskin. “Optimizing QPCR Annealing Temperatures for Cancer-Related LncRNAs”. Clinical and Experimental Health Sciences 15, no. 2 (June 2025): 387-91. https://doi.org/10.33808/clinexphealthsci.1596074.
EndNote Yalçınkaya B, Eskin Z (June 1, 2025) Optimizing qPCR Annealing Temperatures for Cancer-Related lncRNAs. Clinical and Experimental Health Sciences 15 2 387–391.
IEEE B. Yalçınkaya and Z. Eskin, “Optimizing qPCR Annealing Temperatures for Cancer-Related lncRNAs”, Clinical and Experimental Health Sciences, vol. 15, no. 2, pp. 387–391, 2025, doi: 10.33808/clinexphealthsci.1596074.
ISNAD Yalçınkaya, Burhanettin - Eskin, Zeliha. “Optimizing QPCR Annealing Temperatures for Cancer-Related LncRNAs”. Clinical and Experimental Health Sciences 15/2 (June 2025), 387-391. https://doi.org/10.33808/clinexphealthsci.1596074.
JAMA Yalçınkaya B, Eskin Z. Optimizing qPCR Annealing Temperatures for Cancer-Related lncRNAs. Clinical and Experimental Health Sciences. 2025;15:387–391.
MLA Yalçınkaya, Burhanettin and Zeliha Eskin. “Optimizing QPCR Annealing Temperatures for Cancer-Related LncRNAs”. Clinical and Experimental Health Sciences, vol. 15, no. 2, 2025, pp. 387-91, doi:10.33808/clinexphealthsci.1596074.
Vancouver Yalçınkaya B, Eskin Z. Optimizing qPCR Annealing Temperatures for Cancer-Related lncRNAs. Clinical and Experimental Health Sciences. 2025;15(2):387-91.
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