Relative quantification of BCL2 mRNA for diagnostic usage needs stable uncontrolled genes as reference.
Animals
Biomarkers, Tumor
/ blood
Bone Marrow
/ pathology
Cell Line, Tumor
Datasets as Topic
Disease Models, Animal
Feasibility Studies
Gene Expression Regulation, Neoplastic
Genes, Essential
Hematologic Neoplasms
/ blood
Humans
Leukocytes, Mononuclear
Proto-Oncogene Proteins c-bcl-2
/ blood
RNA, Messenger
/ blood
RNA-Seq
/ standards
Real-Time Polymerase Chain Reaction
/ standards
Reference Standards
Journal
PloS one
ISSN: 1932-6203
Titre abrégé: PLoS One
Pays: United States
ID NLM: 101285081
Informations de publication
Date de publication:
2020
2020
Historique:
received:
24
05
2020
accepted:
02
07
2020
entrez:
14
8
2020
pubmed:
14
8
2020
medline:
2
10
2020
Statut:
epublish
Résumé
Dysregulation of BCL2 is a pathophysiology observed in haematological malignancies. For implementation of available treatment-options it is preferred to know the relative quantification of BCL2 mRNA with appropriate reference genes. For the choice of reference genes-(i) Reference Genes were selected by assessing variation of >60,000 genes from 4 RNA-seq datasets of haematological malignancies followed by filtering based on their GO biological process annotations and proximity of their chromosomal locations to known disease translocations. Selected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using geNorm, NormFinder, BestKeeper and RefFinder. (ii) 43 commonly used Reference Genes were obtained from literature through extensive systematic review. Levels of BCL2 mRNA was assessed by qPCR normalized either by novel reference genes from this study or GAPDH, the most cited reference gene in literature and compared. The analysis showed PTCD2, PPP1R3B and FBXW9 to be the most unregulated genes across lymph-nodes, bone marrow and PBMC samples unlike the Reference Genes used in literature. BCL2 mRNA level shows a consistent higher expression in haematological malignancy patients when normalized by these novel Reference Genes as opposed to GAPDH, the most cited Reference Gene. These reference genes should also be applicable in qPCR platforms using Taqman probes and other model systems including cell lines and rodent models. Absence of sample from healthy-normal individual in diagnostic cases call for careful selection of Reference Genes for relative quantification of a biomarker by qPCR.BCL2 can be used as molecular diagnostics only if normalized with a set of reference genes with stable yet low levels of expression across different types of haematological malignancies.
Identifiants
pubmed: 32785215
doi: 10.1371/journal.pone.0236338
pii: PONE-D-20-15589
pmc: PMC7423076
doi:
Substances chimiques
BCL2 protein, human
0
Biomarkers, Tumor
0
Proto-Oncogene Proteins c-bcl-2
0
RNA, Messenger
0
Types de publication
Journal Article
Observational Study
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e0236338Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
Références
Exp Mol Pathol. 2014 Dec;97(3):433-9
pubmed: 25236575
J Biol Chem. 2009 Feb 27;284(9):5876-84
pubmed: 19106097
Plant Mol Biol. 2012 Jan 31;:
pubmed: 22290409
Clin Chem. 2000 Jul;46(7):913-20
pubmed: 10894833
Comput Biol Chem. 2015 Oct;58:19-24
pubmed: 25988219
Sci Rep. 2020 Jun 10;10(1):9411
pubmed: 32523060
Biotechnol Lett. 2004 Mar;26(6):509-15
pubmed: 15127793
RNA Biol. 2013;10(9):1433-8
pubmed: 23635806
Biotechniques. 2005 May;38(5):785-92
pubmed: 15945375
Genes (Basel). 2019 May 17;10(5):
pubmed: 31108950
PeerJ. 2018 Aug 14;6:e5207
pubmed: 30128175
Biochem J. 2008 Nov 15;416(1):e5-6
pubmed: 18939947
Hepatology. 2018 Jun;67(6):2182-2195
pubmed: 29266543
Cancer Res. 2004 Aug 1;64(15):5245-50
pubmed: 15289330
Clin Chem. 2009 Apr;55(4):611-22
pubmed: 19246619
Sci Rep. 2018 Apr 19;8(1):6267
pubmed: 29674626
Adv Biomed Res. 2014 Feb 28;3:85
pubmed: 24761393
Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
pubmed: 12184808
Oncogene. 2013 Aug 22;32(34):4017-27
pubmed: 22964641
Leukemia. 2003 Dec;17(12):2474-86
pubmed: 14562124
Biomed Res Int. 2018 Jun 11;2018:1670328
pubmed: 29992134
Anal Biochem. 2005 Sep 1;344(1):141-3
pubmed: 16054107
PLoS One. 2019 Mar 21;14(3):e0214305
pubmed: 30897165
BMC Mol Biol. 2005 Feb 18;6:4
pubmed: 15720708
BMC Genomics. 2008 Jul 11;9:328
pubmed: 18620571
Leuk Res. 2011 Feb;35(2):256-9
pubmed: 20952062
Front Oncol. 2017 Feb 20;7:22
pubmed: 28265553
BMC Plant Biol. 2019 Sep 14;19(1):405
pubmed: 31521126
Genes Chromosomes Cancer. 2003 Nov;38(3):274-80
pubmed: 14506704
EMBO J. 1994 Feb 1;13(3):683-91
pubmed: 8313913
Cancer Biol Med. 2015 Mar;12(1):10-22
pubmed: 25859407
J Hematol Oncol. 2018 May 11;11(1):65
pubmed: 29747654
BMC Bioinformatics. 2019 May 29;20(Suppl 10):245
pubmed: 31138119
Drug Deliv Transl Res. 2019 Dec;9(6):1027-1042
pubmed: 31115868
Methods Mol Biol. 2014;1087:315-23
pubmed: 24158833
Ann Hum Genet. 2006 Sep;70(Pt 5):587-93
pubmed: 16907705
Ann Oncol. 2019 Sep 1;30(9):1448-1459
pubmed: 31228184
Cancer Genet Cytogenet. 2000 Jul 15;120(2):155-9
pubmed: 10942808
Leukemia. 2017 Sep;31(9):1872-1881
pubmed: 28017967
Haematologica. 2019 Jul;104(7):1417-1421
pubmed: 30523053
Eur Respir J. 2015 Jul;46(1):207-18
pubmed: 25929957
J Agric Food Chem. 2012 Dec 19;60(50):12296-303
pubmed: 23176309
Chem Biol Drug Des. 2017 May;89(5):681-693
pubmed: 27762064
Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10685-9
pubmed: 7938012
PLoS One. 2013 Sep 17;8(9):e72884
pubmed: 24069164
Cells. 2020 Mar 24;9(3):
pubmed: 32213971
Cancer Metastasis Rev. 2013 Jun;32(1-2):63-76
pubmed: 23081702
Haematologica. 2018 May;103(5):849-856
pubmed: 29472356
Int J Oncol. 2018 Mar;52(3):656-678
pubmed: 29286103
Biochem Biophys Res Commun. 2004 Jan 23;313(4):856-62
pubmed: 14706621
Leukemia. 2015 May;29(5):1163-76
pubmed: 25394713
J Hum Genet. 2018 Apr;63(4):431-446
pubmed: 29382920
Nucleic Acids Res. 2010 Jan;38(Database issue):D792-9
pubmed: 19906719
Cell Death Dis. 2019 Feb 21;10(3):177
pubmed: 30792387
Genes Dev. 2004 Nov 1;18(21):2573-80
pubmed: 15520277
Haematologica. 2000 Dec;85(12):1248-54
pubmed: 11114130
Int J Surg. 2010;8(5):336-41
pubmed: 20171303