Design of a new plasma external reference and analysis of nucleic acid detection

Jiaoyang GU; Kai NIU; Yongxin YU; Linpeng ZHENG; Qiao YANG; Luping ZHANG; Jianguo SUN

doi:10.16016/j.1000-5404.201812172

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Design of a new plasma external reference and analysis of nucleic acid detection

Basic Medicine | 更新时间：2026-01-09

- Design of a new plasma external reference and analysis of nucleic acid detection
- Journal of Third Military Medical University Vol. 41, Issue 13, Pages: 1239-1245(2019)
- 作者机构：
  
  400037 重庆，陆军军医大学(第三军医大学)第二附属医院全军肿瘤研究所
- 作者简介：
- 基金信息：
  
  the National Natural Science Foundation for Young Scholars of China(81602688);the Chongqing Natural Science Foundation(CSTC2016jcyjA2013)
- DOI：10.16016/j.1000-5404.201812172
  CLC： R394-33;R394.3;R446.11
- Received：21 December 2018，
  
  Revised：2019-03-14，
  
  Published：15 July 2019
- 稿件说明：
移动端阅览
Jiaoyang GU, Kai NIU, Yongxin YU, et al. Design of a new plasma external reference and analysis of nucleic acid detection[J]. Journal of Third Military Medical University, 2019, 41(13): 1239-1245.
DOI：

Jiaoyang GU, Kai NIU, Yongxin YU, et al. Design of a new plasma external reference and analysis of nucleic acid detection[J]. Journal of Third Military Medical University, 2019, 41(13): 1239-1245. DOI： 10.16016/j.1000-5404.201812172.

摘要

目的

设计新型血浆外参照(plasma external reference，PLACON)，用于人类血浆mRNA、miRNA的检测分析。

方法

从低级别物种秀丽隐杆线虫基因组中选取一段保守序列，利用BLAST检索PLACON序列的特异性，设计成为新型血浆外参照。以恶性肿瘤患者血浆样品及癌细胞株上清液，利用实时荧光定量PCR法，在血浆或细胞上清液中分别以PLACON、β-actin、GAPDH、U6为参照，检测血浆mRNA、miRNA和细胞上清液mRNA并进行对比分析。

结果

BLAST检索出PLACON序列具有较好的特异性，与人类基因组无交叉性。熔解曲线均显示RT-qPCR反应特异性较高，PLAC

ON在血浆中mRNA水平扩增的循环阈值(cycle threshold，CT)均显著低于GAPDH、β-actin(

0.01)，在血浆中miRNA水平扩增的CT值显著低于U6(

0.01)。PLACON在癌细胞上清中进行mRNA扩增实验的验证，得到相似的结果。

结论

PLACON序列特异性好，在血浆中比内参照更有优势，稳定性更高，扩增效率更高，可用于血浆mRNA、miRNA的定量检测。

Abstract

Objective

To design a novel plasma external reference (PLACON) to detect and analyze human plasma mRNA and miRNA.

Methods

A conserved sequence was selected from the genome of the low-level species

Caenorhabditis elegans

. It was designed as a novel plasma external reference and BLAST was used to detect the specificity of PLACON sequences. Plasma samples of patients with malignant tumors and cancer cell supernatants were collected. And PLACON

β-actin

GAPDH

and U6 which were added to the plasma or cell supernatant respectively

were used for amplification comparison to detect plasma mRNA

miRNA and cell supernatant mRNA

by quantitative real-time PCR (RT-qPCR).

Results

BLAST detected that the PLACON sequence had good specificity and no cross-correlation with the human genome. Melting curves showed high specificity in RT-qPCR reaction. The cycle threshold values of mRNA levels were significantly lower in plasma of PLACON group than the GAPDH and β-actin groups (

0.01). The cycle threshold (CT) value of miRNA level amplification in plasma in PLACON group was significantly lower than that in U6 group (

0.01). PLACON was validated in mRNA amplification experiments in cancer cell supernatants

and similar results were obtained.

Conclusion

PLACON sequence is specific

and shows better amplification advantage as internal reference in plasma

with high stability and efficient amplification. It can be used for quantitative detection of plasma mRNA and miRNA.

关键词

Keywords

references

GADKAR V Y, FILION M. New developments in quantitative real-time polymerase chain reaction technology[J]. Curr Issues Mol Biol, 2014, 16: 1-6.

PARK J J, JIN Y B, LEE Y J, et al . KAI1 suppresses HIF-1α and VEGF expression by blocking CDCP1-enhanced Src activation in prostate cancer[J ] . BMC Cancer, 2012, 12: 81. DOI:10.1186/1471-2407-12-81.

CHEN Z, GU S C, TROJANOWICZ B, et al . Down-regulation of TM4SF is associated with the metastatic potential of gastric carcinoma TM4SF members in gastric carcinoma[J ] .World J Surg Oncol, 2011, 9: 43.DOI: 10.1186/1477-7819-9-43.

CHEN X, LIANG H W, GUAN D P, et al . A combination of Let-7d, Let-7g and Let-7i serves as a stable reference for normalization of serum microRNAs[J ] .PLoS ONE, 2013, 8(11): e79652.DOI:10.1371/journal.pone.0079652.

武世伍, 俞岚, 周蕾, 等. β-半乳糖凝集素3和CD82/KAI1蛋白在非小细胞肺癌组织中的表达及其临床意义[J].中华肿瘤杂志, 2013, 35(2):124-128.DOI:10.3760/cma.j.issn.0253-3766.2013.02.010.

WU S W, YU L, ZHOU L, et al . Expression of Gal-3 and CD82/KAI1 proteins in non-small cell lung cancer and their clinical significance[J ] .Chin J Oncol, 2013, 35(2):124- 128.DOI:10.3760/cma.j.issn.0253-3766.2013.02.010.

BECKER C, HAMMERLE-FICKINGER A, RIEDMAIER I, et al . mRNA and microRNA quality control for RT-qPCR analysis[J ] . Methods, 2010, 50(4): 237-243. DOI:10.1016/j.ymeth.2010.01.010.

ZHOU D, TANG W W, LIU X L, et al . Clinical verification of plasma messenger RNA as novel noninvasive biomarker identified through bioinformatics analysis for lung cancer[J ] .Oncotarget, 2017, 8(27): 43978-43989.DOI:10.18632/oncotarget.16701.

SAVELYEVA A V, KULIGINA E V, BARIAKIN D N, et al . Variety of RNAs in peripheral blood cells, plasma, and plasma fractions[J ] .Biomed Res Int, 2017, 2017: 7404912.DOI:10.1155/2017/7404912.

赵莹莹, 邢卉春.循环miRNA检测方法研究进展及其临床应用[J].中华实验和临床感染病杂志(电子版), 2015, 9(1):108-112.DOI:10.3877/cma.j.issn.1674-1358.2015.01.030.

ZHAO Y Y, XING H C.Research progress and clinical application of circulating miRNA detection methods[J].Chin J Exp Clin Infect Dis (Electr Ed), 2015, 9(1):108-112.DOI:10.3877/cma.j.issn.1674-1358.2015.01.030.

WU Q, YANG Y, WU S W, et al . Ev aluation of the correlation of KAI1/CD82, CD44, MMP7 and β-catenin in the prediction of prognosis and metastasis in colorectal carcinoma[J ] .Diagn Pathol, 2015, 10: 176.DOI:10.1186/s13000-015-0411-0.

ROBERTS T C, COENEN-STASS A M, WOOD M J. Assessment of RT-qPCR normalization strategies for accurate quantification of extracellular microRNAs in murine serum[J]. PLoS ONE, 2014, 9(2): e89237. DOI:10.1371/journal.pone.0089237.

ANDERSEN C L, JENSEN J L, ØRNTOFT T F. Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[J]. Cancer Res, 2004, 64(15): 5245-5250. DOI:10.1158/0008-5472.CAN-04-0496.

YE J, JIN C F, LI N, et al . Selection of suitable reference genes for qRT-PCR normalisation under different experimental conditions in Eucommia ulmoides Oliv[J ] . Sci Rep, 2018, 8(1): 15043. DOI:10.1038/s41598-018-33342-w.

ZHAO X L, FU J P, JIANG L T, et al . Transcriptome-based identification of the optimal reference genes as internal controls for quantitative RT-PCR in razor clam (Sinonovacula constricta)[J ] .Genes Genom, 2018, 40(6): 603-613.DOI: 10.1007/s13258-018-0661-9.

DE CÁSSIA-PIRES R, DE MELO M F, BARBOSA R D, et al . Multiplex PCR as a tool for the diagnosis of Leishmania spp. kDNA and the gapdh Housekeeping gene of mammal hosts[J ] . PLoS ONE, 2017, 12(3): e0173922. DOI:10.1371/journal.pone.0173922.

VERES-SZÉKELY A, PAP D, SZIKSZ E, et al . Selective measurement of α smooth muscle actin: why β-actin can not be used as a Housekeeping gene when tissue fibrosis occurs[J ] . BMC Mol Biol, 2017, 18(1): 12. DOI:10.1186/s12867-017-0089-9.

MORI R, WANG Q C, DANENBERG K D, et al . Both beta-actin and GAPDH are useful reference genes for normalization of quantitative RT-PCR in human FFPE tissue samples of prostate cancer[J ] .Prostate, 2008, 68(14): 1555-1560.DOI:10.1002/pros.20815.

SONG J N, BAI Z G, HAN W, et al . Identification of suitable reference genes for qPCR analysis of serum microRNA in gastric cancer patients[J ] .Dig Dis Sci, 2012, 57(4): 897-904.DOI:10.1007/s10620-011-1981-7.

MADADI S, SOLEIMANI M.U6 as a microRNA normalizer in serum of patients with hepatocellular carcinoma[J].Ann Clin Biochem, 2018: 4563218808820.DOI:10.1177/0004563218808820.

PFAFFL M W, TICHOPAD A, PRGOMET C, et al . Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pair-wise correlations[J ] .Biotechnol Lett, 2004, 26(6): 509-515.DOI:10.1023/b:bile.0000019559.84305.47.

BRATTELID T, WINER L H, LEVY F O, et al . Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies[J ] .BMC Mol Biol, 2010, 11: 22.DOI:10.1186/1471-2199-11-22.

PIORKOWSKI G, BARONTI C, DE LAMBALLERIE X, et al . Development of generic Taqman PCR and RT-PCR assays for the detection of DNA and mRNA of β-actin-encoding sequences in a wide range of animal species[J ] .J Virol Methods, 2014, 202: 101-105.DOI: 10.1016/j.jviromet.2014.02.026.

NAKAO R, YAMAMOTO S, YASUMOTO Y, et al . Impact of denervation-induced muscle atrophy on housekeeping gene expression in mice[J ] .Muscle Nerve, 2015, 51(2): 276-281.DOI:10.1002/mus.24310.

WARRINGTON J A, NAIR A, MAHADEVAPPA M, et al . Comparison of human adult and fetal expression and identification of 535 housek eeping/maintenance genes[J ] . Physiol Genom, 2000, 2(3): 143-147. DOI:10.1152/physiolgenomics.2000.2.3.143.

THELLIN O, ZORZI W, LAKAYE B, et al . Housekeeping genes as internal standards: use and limits[J ] . J Biotechnol, 1999, 75(2/3): 291-295.

GARCÍA-REINA A, RODRÍGUEZ-GARCÍA M J, GALIÁN J. Validation of reference genes for quantitative real-time PCR in tiger beetles across sexes, body parts, sexual maturity and immune challenge[J]. Sci Rep, 2018, 8(1): 10743. DOI:10.1038/s41598-018-28978-7.

CORSI A K, WIGHTMAN B, CHALFIE M. A transparent window into biology: A primer on caenorhabditis elegans[J]. Genetics, 2015, 200(2): 387-407. DOI:10.1534/genetics.115.176099.

AMBROS V, RUVKUN G. Recent molecular genetic explorations of caenorhabditis elegans MicroRNAs[J]. Genetics, 2018, 209(3): 651-673. DOI:10.1534/genetics.118.300291.

BARTŁOMIEJ K, MARCIN R. Reference genes in real-time PCR[J].J Appl Genetics, 2013, 54(4): 391-406.DOI:10.1007/s13353-013-0173-x.

BORGHAEI H, PAZ-ARES L, HORN L, et al . Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer[J ] . N Engl J Med, 2015, 373(17): 1627-1639. DOI:10.1056/NEJMoa1507643.

RECK M, RODRÍGUEZ-ABREU D, ROBINSON A G, et al . Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer[J ] . N Engl J Med, 2016, 375(19): 1823-1833. DOI:10.1056/NEJMoa1606774.

ILIÉ M, SZAFER-GLUSMAN E, HOFMAN V, et al . Detection of PD-L1 in circulating tumor cells and white blood cells from patients with advanced non-small-cell lung cancer[J ] . Ann Oncol, 2018, 29(1): 193-199. DOI:10.1093/annonc/mdx636.

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