title: An optimised direct lysis method for gene expression studies on low cell numbers.
abstract: There is increasing interest in gene expression analysis of either single cells or limited numbers of cells. One such application is the analysis of harvested circulating tumour cells (CTCs), which are often present in very low numbers. A highly efficient protocol for RNA extraction, which involves a minimal number of steps to avoid RNA loss, is essential for low input cell numbers. We compared several lysis solutions that enable reverse transcription (RT) to be performed directly on the cell lysate, offering a simple rapid approach to minimise RNA loss for RT. The lysis solutions were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in low cell numbers isolated from four breast cancer cell lines. We found that a lysis solution containing both the non-ionic detergent (IGEPAL CA-630, chemically equivalent to Nonidet P-40 or NP-40) and bovine serum albumin (BSA) gave the best RT-qPCR yield. This direct lysis to reverse transcription protocol outperformed a column-based extraction method using a commercial kit. This study demonstrates a simple, reliable, time- and cost-effective method that can be widely used in any situation where RNA needs to be prepared from low to very low cell numbers.
authors.affiliation: 1] University of Melbourne Department of Surgery, St Vincent's Hospital, Melbourne, Victoria, Australia [2] Translational Genomics &Epigenomics, Olivia Newton-John Cancer Research Institute, Melbourne, Victoria, Australia.
authors.forename: Anh Viet-Phuong
authors.initials: AV
authors.lastname: Le
authors.affiliation: University of Melbourne Department of Surgery, St Vincent's Hospital, Melbourne, Victoria, Australia.
authors.forename: Dexing
authors.initials: D
authors.lastname: Huang
authors.affiliation: University of Melbourne Department of Surgery, St Vincent's Hospital, Melbourne, Victoria, Australia.
authors.forename: Tony
authors.initials: T
authors.lastname: Blick
authors.affiliation: 1] University of Melbourne Department of Surgery, St Vincent's Hospital, Melbourne, Victoria, Australia [2] Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, Brisbane, Queensland, Australia [3] St. Vincent's Institute, Melbourne, Victoria, Australia.
authors.forename: Erik W
authors.initials: EW
authors.lastname: Thompson
authors.affiliation: 1] Translational Genomics &Epigenomics, Olivia Newton-John Cancer Research Institute, Melbourne, Victoria, Australia [2] Department of Pathology, University of Melbourne, Melbourne, Victoria, Australia [3] School of Cancer Medicine, La Trobe University, Melbourne, Victoria, Australia.
authors.forename: Alexander
authors.initials: A
authors.lastname: Dobrovic
mesh_terms: D002458:Cell Fractionation; D045744:Cell Line, Tumor; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D012313:RNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D059467:Transcriptome
chemical_list: D012313:RNA