Intracellular flow cytometry may be combined with good quality and high sensitivity RT-qPCR analysis.
Flow cytometry (FCM) has become a well-established method for analysis of both intracellular and cell-surface proteins, while quantitative RT-PCR (RT-qPCR) is used to determine gene expression with high sensitivity and specificity. Combining these two methods would be of great value. The effects of intracellular staining on RNA integrity and RT-qPCR sensitivity and quality have not, however, been fully examined. We, therefore, intended to assess these effects further. Cells from the human lung cancer cell line A549 were fixed, permeabilized and sorted by FCM. Sorted cells were analyzed using RT-qPCR. RNA integrity was determined by RNA quality indicator analysis. A549 cells were then mixed with cells of the mouse cardiomyocyte cell line HL-1. A549 cells were identified by the cell surface marker ABCG2, while HL-1 cells were identified by intracellular cTnT. Cells were sorted and analyzed by RT-qPCR. Finally, cell cultures from human atrial biopsies were used to evaluate the effects of fixation and permeabilization on RT-qPCR analysis of nonimmortalized cells stored prior to analysis by FCM. A large amount of RNA could be extracted even when cells had been fixed and permeabilized. Permeabilization resulted in increased RNA degradation and a moderate decrease in RT-qPCR sensitivity. Gene expression levels were also affected to a moderate extent. Sorted populations from the mixed A549 and HL-1 cell samples showed gene expression patterns that corresponded to FCM data. When samples were stored before FCM sorting, the RT-qPCR analysis could still be performed with high sensitivity and quality. In summary, our results show that intracellular FCM may be performed with only minor impairment of the RT-qPCR sensitivity and quality when analyzing sorted cells; however, these effects should be considered when comparing RT-qPCR data of not fixed samples with those of fixed and permeabilized samples.
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.|Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden.
Mikael
M
Sandstedt
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.|Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden.
Marianne
M
Jonsson
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.|Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden.
Julia
J
Asp
Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.|Department of Cardiothoracic Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden.
Göran
G
Dellgren
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.|Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden.
Anders
A
Lindahl
Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.|Department of Cardiothoracic Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden.
Anders
A
Jeppsson
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.|Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden.
Joakim
J
Sandstedt
D000818:Animals; D001706:Biopsy; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D005434:Flow Cytometry / Q000379:methods*; D005786:Gene Expression Regulation; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D017493:Leukocyte Common Antigens / Q000378:metabolism; D051379:Mice; D009206:Myocardium / Q000166:cytology; D011309:Preservation, Biological; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012680:Sensitivity and Specificity; D016707:Tissue Fixation
D012313:RNA; D019009:Proto-Oncogene Proteins c-kit; D017493:Leukocyte Common Antigens
FACS; RNA degradation; RNA integrity; RNA isolation; cardiac progenitor cells; fixation; flow cytometry; intracellular staining; permeabilization; quantitative real time PCR