title: Direct quantification of mRNA and miRNA from cell lysates using reverse transcription real time PCR: a multidimensional analysis of the performance of reagents and workflows.
abstract: Substantial efforts have been devoted to in vitro testing of candidate chemotherapeutics by profiling transcriptional changes across the collection of NCI-60 cell-lines. A work-flow with reagents that enable the direct quantification of RNA of different molecular sizes simultaneously in the same sample without laborious total RNA isolation will invariably increase the throughput and accuracy of the study. MicroRNAs (miRNAs) are known to regulate most cellular functions, acting post-transcriptionally by repressing numerous eukaryotic mRNAs. Recent findings on the remarkable stability of miRNA prompted us to investigate the feasibility of quantifying the expression levels of both mRNA and miRNA directly from cell lysates (cell-to-Ct). Multidimensional analyses of the expressions of mRNA and miRNA across seven NCI-60 cell lines and multiple reagents were conducted to assess the performances of these reagents and workflows for cell-to-Ct measurements using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Quantification of RNA species using lysates prepared from an in-house and one of the commercial reagents demonstrated comparable performance to those prepared by the more laborious and conventional method of using guanidinium-phenol-chloroform. Additionally, miRNA was found to be highly stable in the cell lysates when incubated at room temperature for prolonged period of time and subjected to multiple freeze-thaw cycles. In summary, this study demonstrated significant differences in pre-analytical performance of a variety of commercially available reagents and described a cost-effective reagent useful for rapid, scalable, and high-throughput workflow for the detection of mRNA and miRNA from the same biological sample.
authors.affiliation: Department of Biochemistry, National University of Singapore, Singapore, Singapore.
authors.forename: Yoon Khei
authors.initials: YK
authors.lastname: Ho
authors.forename: Wen Ting
authors.initials: WT
authors.lastname: Xu
authors.forename: Heng Phon
authors.initials: HP
authors.lastname: Too
mesh_terms: D002457:Cell Extracts / Q000737:chemistry; D002458:Cell Fractionation; D045744:Cell Line, Tumor; D015925:Cryopreservation; D020869:Gene Expression Profiling; D006146:Guanidines / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010636:Phenols / Q000737:chemistry; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012996:Solutions
chemical_list: D002457:Cell Extracts; D006146:Guanidines; D035683:MicroRNAs; D010636:Phenols; D012333:RNA, Messenger; D012996:Solutions; C411644:trizol
references.citation: Mol Cancer Ther. 2010 May;9(5):1080-91
references.pmid: 20442302
references.citation: EMBO Mol Med. 2013 Jul;5(7):1051-66
references.pmid: 23666744
references.citation: Diagn Mol Pathol. 2007 Mar;16(1):38-49
references.pmid: 17471157
references.citation: Cancer Res. 2006 Aug 1;66(15):7390-4
references.pmid: 16885332
references.citation: Cancer Res. 2007 Mar 15;67(6):2456-68
references.pmid: 17363563
references.citation: Cancer Res. 2010 Mar 1;70(5):1753-8
references.pmid: 20160033
references.citation: Biol Psychiatry. 1993 Mar 15;33(6):456-66
references.pmid: 8098224
references.citation: J Vis Exp. 2011 Aug 03;(54):
references.pmid: 21847076
references.citation: Biochem Biophys Res Commun. 2007 Feb 23;353(4):1052-5
references.pmid: 17204243
references.citation: Science. 2006 Sep 29;313(5795):1929-35
references.pmid: 17008526
references.citation: Clin Chem. 2010 Jun;56(6):998-1006
references.pmid: 20378769
references.citation: Genome Res. 2009 Jan;19(1):92-105
references.pmid: 18955434
references.citation: Nat Protoc. 2009;4(1):37-43
references.pmid: 19131955
references.citation: BMC Genomics. 2010 Jan 29;11:75
references.pmid: 20113474
references.citation: Oncogene. 2001 Sep 27;20(43):6263-8
references.pmid: 11593437
references.citation: Diagn Mol Pathol. 2009 Mar;18(1):44-52
references.pmid: 19214109
references.citation: Clin Chem. 2009 Sep;55(9):1719-27
references.pmid: 19617290
references.citation: Diagn Pathol. 2009 Mar 24;4:9
references.pmid: 19317905
references.citation: Leukemia. 2006 Aug;20(8):1385-92
references.pmid: 16761018
references.citation: RNA. 2010 Jul;16(7):1436-45
references.pmid: 20547774
references.citation: Nature. 2010 Aug 12;466(7308):835-40
references.pmid: 20703300
references.citation: Annu Rev Genomics Hum Genet. 2008;9:387-402
references.pmid: 18576944
references.citation: J Clin Pathol. 2009 May;62(5):422-8
references.pmid: 19398594
references.citation: Pharmacogenomics J. 2007 Jun;7(3):212-20
references.pmid: 16940966
references.citation: Brain Res Mol Brain Res. 2003 Jun 10;114(2):146-53
references.pmid: 12829325
references.citation: Nat Genet. 2000 Mar;24(3):236-44
references.pmid: 10700175
references.citation: Biochim Biophys Acta. 2008 Nov;1779(11):758-65
references.pmid: 18439437
references.citation: Mol Cancer Ther. 2010 Dec;9(12):3105-14
references.pmid: 21159603
references.citation: BMC Biotechnol. 2010 Sep 06;10:64
references.pmid: 20815927
references.citation: Br J Cancer. 2007;96 Suppl:R40-4
references.pmid: 17393584
references.citation: Nat Rev Cancer. 2006 Oct;6(10):813-23
references.pmid: 16990858
references.citation: Mol Cancer Ther. 2007 May;6(5):1483-91
references.pmid: 17483436
references.citation: Nat Genet. 2002 Dec;32 Suppl:509-14
references.pmid: 12454646
references.citation: Biochem Biophys Res Commun. 2009 Dec 4;390(1):1-4
references.pmid: 19769940
references.citation: Biochim Biophys Acta. 2008 Nov;1779(11):749-57
references.pmid: 18316046
references.citation: RNA. 2007 Oct;13(10):1668-74
references.pmid: 17698639
references.citation: Annu Rev Genomics Hum Genet. 2009;10:135-51
references.pmid: 19715439
references.citation: RNA. 2010 May;16(5):991-1006
references.pmid: 20360395
references.citation: Nat Rev Cancer. 2006 Nov;6(11):857-66
references.pmid: 17060945