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Messenger RNA quantification after fluorescence activated cell sorting using intracellular antigens
Biochemical and Biophysical Research Communications 397 (2010) 425–428
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Messenger RNA quantification after fluorescence activated cell sorting using intracellular antigens Hiroya Yamada a,b, Rie Maruo a,b, Mikio Watanabe b, Yoh Hidaka a, Yoshinori Iwatani b, Toru Takano a,* a b
Department of Laboratory Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan Division of Health Sciences, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
a r t i c l e
i n f o
Article history: Received 11 May 2010 Available online 27 May 2010 Keywords: Flow cytometry Cell sorting RT-PCR Stem cell Cancer stem cell FACS
a b s t r a c t Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. The only possible method for carrying out such procedures is fluorescence activated cell sorting (FACS). However, FACS has the following limitations. First, cells without an appropriate cell surface marker cannot be sorted. Second, the cells have to be kept alive during the sorting process in order to analyze their biological characteristics. If an intracellular antigen that was specific to a particular cell type could be stained with a florescent dye and then the cells can be sorted without causing RNA degradation, a more simple and universal method for sorting and analyzing cells with a specific gene expression pattern could be established since the biological characteristics of the sorted cells could then be determined by analyzing their gene expression profile. In this study, we established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) targeting intracellular antigens. This method can be used for the detection and analysis of stem cells or cancer stem cells in various tissues. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Stem cells have the potential to indefinitely self renew and differentiate into the various cell types of a particular tissue [1]. Their differentiation potential and capacity for tissue renewal and damage repair make stem cells valuable for regenerative medicine, tissue engineering, and biotechnology applications [2]. On the other hand, a series of mutations affecting the differentiation potential of stem cell and their unlimited growth potential may generate cancer stem cells (CSCs) [3–5]. There is increasing evidence that CSCs play an important role in the biological behavior of tumors, and they may even determine patient prognosis [6–10]. Stem cells and CSCs usually exist as minor populations of cells in a tissue [11]. They can be separated from other cells by fluorescent activated cell sorting (FACS) using antibodies that specifically bind to cell surface marker proteins or by the transfection of a plasmid carrying a cell type specific promoter and a reporter gene [6–12]. However, the majority of stem cells and CSCs do not possess such antigens or do not show specific promoter activity and so cannot be sorted in this manner. Furthermore, cell samples must be treated under sterilized conditions and kept alive throughout the whole procedure since the sorted cells need to be cultured in order to have their biological characteristics determined. In
* Corresponding author. Fax: +81 6 6879 6635. E-mail address: [email protected] (T. Takano). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.05.112
addition, transfection of a foreign reporter gene may result in the modification of the properties of the cell. Many reports have described the sorting of various types of cells using intracellular protein markers and FACS [13–15]. However, few studies have then subjected the sorting cells to molecular analysis since the fixation and permeabilization required for the staining of intracellular antigens, especially nuclear antigens, often cause RNA degradation [16–19]. Thus, a simpler method for sorting and analyzing stem cells needs to be established. If cellular RNA could be preserved during the staining of intracellular antigenes, then the stained cells could be sorted by FACS, and their biological characteristics could be analyzed by obtaining their gene expression profile. In this study, we developed an in-tube fluorescence immunocytochemistry method for FACS that minimizes the degradation of cellular RNA and established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) targeting intracellular antigens. 2. Materials and methods 2.1. Cell culture Three cell lines were used in this study: the rat thyroid cell line FRTL-5, which expresses thyroglobulin (TG) and thyroid transcriptional factor 1 (TTF-1); the human lung carcinoma cell line PC3, which expresses TTF-1; and the human anaplastic thyroid
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carcinoma cell line 8305C, which does not express either of these genes. The FRTL-5 cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and the PC3 and 8305C cells were provided by the Human Science Research Resource Bank (Osaka, Japan). The FRTL-5 cells were cultured in Ham’s F-12 medium (Gibco, Paisley, Scotland, UK), supplemented with 5% newborn calf serum (Gibco); 2.5 mg/ml sodium bicarbonate (NaHCO3) (Wako, Osaka, Japan); 100 U/ml penicillin (Meiji, Tokyo, Japan); 100 lg/ml streptomycin (Meiji); 250 ng/ml fungizone (Gibco); and a six-hormone preparation consisting of 100 lU/ml TSH (Sigma, St. Louis, MO, USA), 10 lg/ml bovine insulin (Sigma), 10 ng/ml somatostatin (Sigma), 360 pg/ml hydrocortisone (Sigma), 5 lg/ml transferrin (Gibco), and 100 ng/ml glycyl-L-histidyl-L-lysine acetate (Sigma). The PC3 and 8305C cells were grown in RPMI 1640 medium (Gibco), supplemented with 10% fetal bovine serum (Gibco), 2 mg/ml NaHCO3, 100 U/ml penicillin, 75 lg/ml streptomycin, and 250 ng/ml fungizone. All cells were maintained in a 5% CO2–95% air atmosphere at 37 °C, and the medium was changed every third day until the cells were subconfluent. 2.2. In-tube immunocytochemistry for FACS Ten ml tubes (Azone, Osaka, Japan) were used in all procedures. All media and buffers except the antibodies were mixed with 0.1% diethylpyrocarbonate (DEPC) (Sigma), stored overnight at room temperature, and then autoclaved. The FRTL-5, PC3, and 8305C cells were fixed with methanol (Wako) supplemented with 10% polyethylene glycols (molecular weight 300 kD, Wako) (UM-Fix) for 15 min at 4 °C. UM-Fix was replaced by phosphate-buffered saline (PBS) (Wako) supplemented with 0.1% Tween 20 (PBS-T) (Sigma) and kept in PBS containing 10% dimethyl sulfoxide (DMSO) (Sigma) at 80 °C until use. On the day of immunocytochemistry, 1 106 cells were thawed rapidly in a water bath at 37 °C and then permeabilized with 0.1% TritonX-100 (Sigma) in PBS for 30 min at 4 °C. The cells were washed with PBS-T and then blocked with 0.5% blocking reagent (Roche Diagnostics) in PBS-T (blocking solution). The blocked cells were then incubated in 100 ll of fluoresceinconjugated mouse anti-thyroglobulin antibody (No. 53905, AnaSpec, San Jose, CA, USA) and R-phycoerythrin (R-PE)-conjugated TTF-1 antibody (DAKO, Carpinteria, CA Clone AG7G3/1) diluted 200 times in the blocking solution overnight at 4 °C. The antibodies were labeled with fluorescein and R-PE using the Fluorescein and R-PE Labeling Kit-NH2 (Dojindo Molecular Technologies, Kumamoto, Japan), respectively. After being washed twice with PBS-T, the cells were analyzed using a flow cytometer.
using SYBRÒ Green Master Mix and the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City CA, USA) according to the manufacturer’s recommendations. One microliter of cDNA was used in the subsequent assays. The two primers used for the quantification of rat ACTB cDNA were as follows: rACTBF (0.5 lM): 50 -CTGACAGGATGCAGAAGGA-30 ; rACTBR (0.5 lM): 50 -TGATCCACATCTGCTGGAA-30 . Those used for human ACTB cDNA were as follows: hACTBF (0.5 lM): 50 -TGGACATCCGCAAAGACCTG-30 ; rACTBR (0.5 lM): 50 -CCGATCCACACGGAGTACTT-30 . Those used for rat TG cDNA were as follows: rTGF (0.5 lM): 50 -ATGGCCAGTACCTACGTGAA-30 ; rTGR (0.5 lM): 50 -CCTTTGCCCTGTTGATAAGCC-30 . Those used for human SFTPB cDNA were as follows: hSFTBPF (0.5 lM): 50 -AGCTTTCTTCCTCGAGATG-30 ; hSFTPBR (0.5 lM): 50 -CACAGCAGAAATAGAATCACC-30 . All primers were purchased from Operon Biotechnologies (Tokyo, Japan). The conditions for PCR were as follows: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. A recombinant pGEM T-vector (Promega, Tokyo, Japan) containing a partial cDNA was constructed by PCR-cloning with the same primer sets as used in the PCR and was used as a standard sample. 2.5. Flow cytometry and data analysis Flow cytometric analysis and sorting were performed using a FACS Vantage SE from Becton Dickinson (Mountain View, CA, USA) equipped with a 488-nm argon ion laser and emission filters for fluorescein and R-PE. For each sample, at least 104 events were collected and analyzed. Data were analyzed using the DiVa software (Becton Dickinson). 104 cells with strong fluorescent signals were sorted into TG positive, and TTF-1 positive and TG negative fractions. The sorted cells were stored in RNA later (Applied Biosystems). 3. Results 3.1. Cell preservation at 80 °C after fixation After fixation, the cells were stored at 80 °C in PBS supplemented with 10% DMSO. The copy number of human ACTB mRNA was measured in 106 8305C cells stored for a period ranging from 1 day to 2 weeks. No significant decrease in the copy number was observed in the cells stored for 2 weeks (Fig. 1). In addition, no change in cell morphology was observed (data not shown).
2.3. Extraction of RNA from the cells and reverse transcription (RT) Total RNA were extracted from the cells as described previously [20]. The recovered total RNA was reverse transcribed in an RT mixture, which contained 4 ll of 5 first strand buffer (Invitrogen, Tokyo, Japan), 10 mmol/l dithiothreitol (Invitrogen), 0.5 mmol/l deoxynucleotide triphosphates (dNTP) (Takara, Shiga, Japan), 200 U Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen), 2 U/ll RNase inhibitor (Takara), and 2.5 lmol/l random hexamer (Takara) in a total volume of 20 ll. The RT reaction was carried out for 10 min at 25 °C, 50 min at 42 °C, and 15 min at 70 °C. 2.4. Real-time quantitative polymerase chain reaction The real-time quantitative polymerase chain reactions (PCR) for rat b-actin (ACTB), human ACTB, rat TG, and human pulmonaryassociated surfactant B protein (SFTPB) mRNA were performed
3.2. The copy number of ACTB mRNA before and after immunocytochemistry We measured the copy number of human ACTB mRNA in 106 8305C cells before and after immunocytochemistry. No significant decrease was observed after the immunocytochemistry procedure (Fig. 2). 3.3. Quantification of mRNA after FACS After immunocytochemistry, the FRTL-5, PC3, and 8305C cells were analyzed and sorted by flow cytometry (Fig. 3). When double stained with fluorescein-labeled TG antibody and R-PE-labeled TTF-1 antibody, the FRTL-5 cells, which are known to express TG and TTF-1, showed a clear shift to the right and small shift to the upper. The PC3 cells, which are known to express TTF-1 but not TG, showed a clear upwards shift.
H. Yamada et al. / Biochemical and Biophysical Research Communications 397 (2010) 425–428
Fig. 1. The copy number of ACTB mRNA in fixed cells stored at 80 °C. After fixation with UM-Fix, the cells were preserved in PBS supplemented with 10% DMSO at 80 °C for up to 2 weeks. Then, 106 cells were collected by centrifugation, and total RNA was extracted. The copy number of human ACTB mRNA was measured by realtime quantitative PCR. The copy number of human ACTB mRNA immediately after fixation was designated as 1.0. The results are shown as the means ± SD of triplicate determinations.
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Fig. 2. The copy number of ACTB mRNA in 8305C cells before and after immunocytochemistry. 106 8305C cells were collected before and after immunocytochemistry, and then total RNA was extracted. The copy number of human ACTB mRNA was measured by real-time quantitative PCR. The mean copy number of human ACTB mRNA before staining was designated as 1.0. The results are shown as the means ± SD of triplicate determinations.
as expected, the FRTL-5 and PC3 cells had been collected as TG positive, and TTF-1 positive and TG negative fractions, respectively. The three cell lines were then mixed and sorted into two fractions: the TG positive fraction corresponding to FRTL-5 cells and the TG negative and TTF-1 positive fraction corresponding to PC3 cells. 104 cells were collected in each fraction, and then the copy numbers of rat ACTB, rat TG, human ACTB, and human SFTBP mRNA in each fraction were measured (Table 1). In the TG positive fraction, rat ACTB and TG mRNA in all cases, but not human ACTB or SFTBP mRNA were detected. On the other hand, in the TTF-1 positive and TG negative fraction, human ACTB and SFTBP mRNA, but not rat ACTB or TG mRNA were detected, which suggested that,
4. Discussion In this study, we established a novel in-tube immunocytochemistry method that preserved cellular RNA even after staining, which made it possible to separate the stained cells by FACS. Thus, using this method any cells of interest can be separated by FACS using intracellular and/or nuclear antigens as markers, and then their biological characteristics can be analyzed by extracting their RNA and comparing their gene expression profiles.
Fig. 3. Flow cytometry and cell sorting. After immunocytochemistry, the FRTL-5, PC3, and 8305C cells were analyzed and sorted by flow cytometry with fluorescein-labeled TG antibody and R-PE labeled TTF-1 antibody. A: 8305C cells; B: FRTL-5 cells; C: PC3 cells; D: a mixture of 8305C, FRTL-5, and PC3 cells. (a) shows the gating range of the TG positive fraction (right quadrant), and (b) shows that of the TTF-1 positive and TG negative fraction (upper left quadrant).
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Table 1 Quantitative RT-PCR analysis of the cells before and after sorting. mRNA copy 104
TG positive cell
TTF-1 positive and TG negative cells
8305 cells before sorting
FRTL-5 cells before sorting
PC3 cells before sorting
Rat ACTB mRNA Human ACTB mRNA Rat TG mRNA Human SFTBP mRNA Rat TG/rat ACTB mRNA Human SFTBP/human ACTB mRNA
3.6 ± 0.1 N.D. 174.4 ± 31.1 N.D. 47.6 ± 8.1 —
N.D. 13.0 ± 2.0 N.D. 14.6 ± 1.8 — 1.01 ± 0.2
N.D. 3.6 ± 0.8 N.D. N.D. — —
6.0 ± 0.8 N.D. 166.6 ± 25.1 N.D. 28.2 ± 7.9 —
N.D. 9.0 ± 0.5 N.D. 11.6 ± 3.8 — 1.31 ± 0.5
104 stained cells were collected before and after sorting, and then their copy numbers of rat ACTB, ratTG, human ACTB, and SFTBP mRNAs were measured. The results are shown as the means ± SD of triplicate determinations. N.D., not detected.
Several studies have reported on the staining of intracellular antigens of fixed cells in suspension and the subsequent detection of the stained cells by FACS [13–16]. However, these studies did not confirm whether quantitative measurement of mRNA can be carried out after staining. When we started this project, the number of cells was greatly decreased after the staining procedure since fixed cells are sticky and so adhere to the plastic tube. We found that adding 0.1% Tween 20 to all buffers markedly increased the cell number after each washing step (data not shown). We also found that methanol supplemented with PEG was a suitable fixative for preserving RNA and cell morphology during staining, and we treated all media except those containing antibodies with DEPC as we found that use of the blocking reagent without DEPC treatment resulted in cellular RNA degradation (data not shown). Cells fixed in this manner can be stored at 80 °C for at least 2 weeks without changes in their cell morphology or the copy number of cellular RNA occurring. TG is one of the most abundantly expressed genes in differentiated thyroid cells [20,21], and TTF-1 is expressed in lung and thyroid cells [21,22]. We used FRTL-5 cells, a rat thyroid cell line, as TG and TTF-1 positive cells and PC3 cells, a human lung carcinoma cell line, as TTF-1 positive and TG negative cells. 8305C cells, which are derived from an undifferentiated thyroid carcinoma, do not express TG or TTF-1. The results regarding the gene expression levels of the cells in the TG positive fraction and the TTF-1 positive and TG negative fraction were quite reasonable. Rat ACTB and rat TG mRNA were detected in the TG positive fraction, which mainly contained FRTL-5 cells but did not containing 8305C or PC3 cells. Also, human ACTB and SFTBP mRNAs were detected in the TTF-1 positive and TG negative fraction, which mainly contained PC3 cells but did not containing FRTL-5 or 8305C cells. These results indicated that the gene expression profiles of cell fractions sorted by FACS are reliable and can be used to identify the biological characteristics of the targeted cells. 5. Conclusions We succeeded in establishing a novel immunocytochemistry method for mRNA quantification after FACS. These protocols will hopefully contribute to the accumulation of knowledge regarding human stem cells and CSCs, the biological characteristics of which are mostly unknown. Acknowledgments This research was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan via a Grant-in-Aid for Scientific Research C, 2008–2010, No. 20590570; a Research Grant from the Princess Takamatsu Cancer Research Fund 04-23606; and the Japanese Society of Laboratory Medicine Fund for the Promotion of Scientific Research.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Messenger RNA quantification after fluorescence activated cell sorting using intracellular antigens Hiroya Yamada a,b, Rie Maruo a,b, Mikio Watanabe b, Yoh Hidaka a, Yoshinori Iwatani b, Toru Takano a,* a b
Department of Laboratory Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan Division of Health Sciences, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan
a r t i c l e
i n f o
Article history: Received 11 May 2010 Available online 27 May 2010 Keywords: Flow cytometry Cell sorting RT-PCR Stem cell Cancer stem cell FACS
a b s t r a c t Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. The only possible method for carrying out such procedures is fluorescence activated cell sorting (FACS). However, FACS has the following limitations. First, cells without an appropriate cell surface marker cannot be sorted. Second, the cells have to be kept alive during the sorting process in order to analyze their biological characteristics. If an intracellular antigen that was specific to a particular cell type could be stained with a florescent dye and then the cells can be sorted without causing RNA degradation, a more simple and universal method for sorting and analyzing cells with a specific gene expression pattern could be established since the biological characteristics of the sorted cells could then be determined by analyzing their gene expression profile. In this study, we established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) targeting intracellular antigens. This method can be used for the detection and analysis of stem cells or cancer stem cells in various tissues. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Stem cells have the potential to indefinitely self renew and differentiate into the various cell types of a particular tissue [1]. Their differentiation potential and capacity for tissue renewal and damage repair make stem cells valuable for regenerative medicine, tissue engineering, and biotechnology applications [2]. On the other hand, a series of mutations affecting the differentiation potential of stem cell and their unlimited growth potential may generate cancer stem cells (CSCs) [3–5]. There is increasing evidence that CSCs play an important role in the biological behavior of tumors, and they may even determine patient prognosis [6–10]. Stem cells and CSCs usually exist as minor populations of cells in a tissue [11]. They can be separated from other cells by fluorescent activated cell sorting (FACS) using antibodies that specifically bind to cell surface marker proteins or by the transfection of a plasmid carrying a cell type specific promoter and a reporter gene [6–12]. However, the majority of stem cells and CSCs do not possess such antigens or do not show specific promoter activity and so cannot be sorted in this manner. Furthermore, cell samples must be treated under sterilized conditions and kept alive throughout the whole procedure since the sorted cells need to be cultured in order to have their biological characteristics determined. In
* Corresponding author. Fax: +81 6 6879 6635. E-mail address: [email protected] (T. Takano). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.05.112
addition, transfection of a foreign reporter gene may result in the modification of the properties of the cell. Many reports have described the sorting of various types of cells using intracellular protein markers and FACS [13–15]. However, few studies have then subjected the sorting cells to molecular analysis since the fixation and permeabilization required for the staining of intracellular antigens, especially nuclear antigens, often cause RNA degradation [16–19]. Thus, a simpler method for sorting and analyzing stem cells needs to be established. If cellular RNA could be preserved during the staining of intracellular antigenes, then the stained cells could be sorted by FACS, and their biological characteristics could be analyzed by obtaining their gene expression profile. In this study, we developed an in-tube fluorescence immunocytochemistry method for FACS that minimizes the degradation of cellular RNA and established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) targeting intracellular antigens. 2. Materials and methods 2.1. Cell culture Three cell lines were used in this study: the rat thyroid cell line FRTL-5, which expresses thyroglobulin (TG) and thyroid transcriptional factor 1 (TTF-1); the human lung carcinoma cell line PC3, which expresses TTF-1; and the human anaplastic thyroid
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carcinoma cell line 8305C, which does not express either of these genes. The FRTL-5 cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and the PC3 and 8305C cells were provided by the Human Science Research Resource Bank (Osaka, Japan). The FRTL-5 cells were cultured in Ham’s F-12 medium (Gibco, Paisley, Scotland, UK), supplemented with 5% newborn calf serum (Gibco); 2.5 mg/ml sodium bicarbonate (NaHCO3) (Wako, Osaka, Japan); 100 U/ml penicillin (Meiji, Tokyo, Japan); 100 lg/ml streptomycin (Meiji); 250 ng/ml fungizone (Gibco); and a six-hormone preparation consisting of 100 lU/ml TSH (Sigma, St. Louis, MO, USA), 10 lg/ml bovine insulin (Sigma), 10 ng/ml somatostatin (Sigma), 360 pg/ml hydrocortisone (Sigma), 5 lg/ml transferrin (Gibco), and 100 ng/ml glycyl-L-histidyl-L-lysine acetate (Sigma). The PC3 and 8305C cells were grown in RPMI 1640 medium (Gibco), supplemented with 10% fetal bovine serum (Gibco), 2 mg/ml NaHCO3, 100 U/ml penicillin, 75 lg/ml streptomycin, and 250 ng/ml fungizone. All cells were maintained in a 5% CO2–95% air atmosphere at 37 °C, and the medium was changed every third day until the cells were subconfluent. 2.2. In-tube immunocytochemistry for FACS Ten ml tubes (Azone, Osaka, Japan) were used in all procedures. All media and buffers except the antibodies were mixed with 0.1% diethylpyrocarbonate (DEPC) (Sigma), stored overnight at room temperature, and then autoclaved. The FRTL-5, PC3, and 8305C cells were fixed with methanol (Wako) supplemented with 10% polyethylene glycols (molecular weight 300 kD, Wako) (UM-Fix) for 15 min at 4 °C. UM-Fix was replaced by phosphate-buffered saline (PBS) (Wako) supplemented with 0.1% Tween 20 (PBS-T) (Sigma) and kept in PBS containing 10% dimethyl sulfoxide (DMSO) (Sigma) at 80 °C until use. On the day of immunocytochemistry, 1 106 cells were thawed rapidly in a water bath at 37 °C and then permeabilized with 0.1% TritonX-100 (Sigma) in PBS for 30 min at 4 °C. The cells were washed with PBS-T and then blocked with 0.5% blocking reagent (Roche Diagnostics) in PBS-T (blocking solution). The blocked cells were then incubated in 100 ll of fluoresceinconjugated mouse anti-thyroglobulin antibody (No. 53905, AnaSpec, San Jose, CA, USA) and R-phycoerythrin (R-PE)-conjugated TTF-1 antibody (DAKO, Carpinteria, CA Clone AG7G3/1) diluted 200 times in the blocking solution overnight at 4 °C. The antibodies were labeled with fluorescein and R-PE using the Fluorescein and R-PE Labeling Kit-NH2 (Dojindo Molecular Technologies, Kumamoto, Japan), respectively. After being washed twice with PBS-T, the cells were analyzed using a flow cytometer.
using SYBRÒ Green Master Mix and the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City CA, USA) according to the manufacturer’s recommendations. One microliter of cDNA was used in the subsequent assays. The two primers used for the quantification of rat ACTB cDNA were as follows: rACTBF (0.5 lM): 50 -CTGACAGGATGCAGAAGGA-30 ; rACTBR (0.5 lM): 50 -TGATCCACATCTGCTGGAA-30 . Those used for human ACTB cDNA were as follows: hACTBF (0.5 lM): 50 -TGGACATCCGCAAAGACCTG-30 ; rACTBR (0.5 lM): 50 -CCGATCCACACGGAGTACTT-30 . Those used for rat TG cDNA were as follows: rTGF (0.5 lM): 50 -ATGGCCAGTACCTACGTGAA-30 ; rTGR (0.5 lM): 50 -CCTTTGCCCTGTTGATAAGCC-30 . Those used for human SFTPB cDNA were as follows: hSFTBPF (0.5 lM): 50 -AGCTTTCTTCCTCGAGATG-30 ; hSFTPBR (0.5 lM): 50 -CACAGCAGAAATAGAATCACC-30 . All primers were purchased from Operon Biotechnologies (Tokyo, Japan). The conditions for PCR were as follows: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. A recombinant pGEM T-vector (Promega, Tokyo, Japan) containing a partial cDNA was constructed by PCR-cloning with the same primer sets as used in the PCR and was used as a standard sample. 2.5. Flow cytometry and data analysis Flow cytometric analysis and sorting were performed using a FACS Vantage SE from Becton Dickinson (Mountain View, CA, USA) equipped with a 488-nm argon ion laser and emission filters for fluorescein and R-PE. For each sample, at least 104 events were collected and analyzed. Data were analyzed using the DiVa software (Becton Dickinson). 104 cells with strong fluorescent signals were sorted into TG positive, and TTF-1 positive and TG negative fractions. The sorted cells were stored in RNA later (Applied Biosystems). 3. Results 3.1. Cell preservation at 80 °C after fixation After fixation, the cells were stored at 80 °C in PBS supplemented with 10% DMSO. The copy number of human ACTB mRNA was measured in 106 8305C cells stored for a period ranging from 1 day to 2 weeks. No significant decrease in the copy number was observed in the cells stored for 2 weeks (Fig. 1). In addition, no change in cell morphology was observed (data not shown).
2.3. Extraction of RNA from the cells and reverse transcription (RT) Total RNA were extracted from the cells as described previously [20]. The recovered total RNA was reverse transcribed in an RT mixture, which contained 4 ll of 5 first strand buffer (Invitrogen, Tokyo, Japan), 10 mmol/l dithiothreitol (Invitrogen), 0.5 mmol/l deoxynucleotide triphosphates (dNTP) (Takara, Shiga, Japan), 200 U Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen), 2 U/ll RNase inhibitor (Takara), and 2.5 lmol/l random hexamer (Takara) in a total volume of 20 ll. The RT reaction was carried out for 10 min at 25 °C, 50 min at 42 °C, and 15 min at 70 °C. 2.4. Real-time quantitative polymerase chain reaction The real-time quantitative polymerase chain reactions (PCR) for rat b-actin (ACTB), human ACTB, rat TG, and human pulmonaryassociated surfactant B protein (SFTPB) mRNA were performed
3.2. The copy number of ACTB mRNA before and after immunocytochemistry We measured the copy number of human ACTB mRNA in 106 8305C cells before and after immunocytochemistry. No significant decrease was observed after the immunocytochemistry procedure (Fig. 2). 3.3. Quantification of mRNA after FACS After immunocytochemistry, the FRTL-5, PC3, and 8305C cells were analyzed and sorted by flow cytometry (Fig. 3). When double stained with fluorescein-labeled TG antibody and R-PE-labeled TTF-1 antibody, the FRTL-5 cells, which are known to express TG and TTF-1, showed a clear shift to the right and small shift to the upper. The PC3 cells, which are known to express TTF-1 but not TG, showed a clear upwards shift.
H. Yamada et al. / Biochemical and Biophysical Research Communications 397 (2010) 425–428
Fig. 1. The copy number of ACTB mRNA in fixed cells stored at 80 °C. After fixation with UM-Fix, the cells were preserved in PBS supplemented with 10% DMSO at 80 °C for up to 2 weeks. Then, 106 cells were collected by centrifugation, and total RNA was extracted. The copy number of human ACTB mRNA was measured by realtime quantitative PCR. The copy number of human ACTB mRNA immediately after fixation was designated as 1.0. The results are shown as the means ± SD of triplicate determinations.
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Fig. 2. The copy number of ACTB mRNA in 8305C cells before and after immunocytochemistry. 106 8305C cells were collected before and after immunocytochemistry, and then total RNA was extracted. The copy number of human ACTB mRNA was measured by real-time quantitative PCR. The mean copy number of human ACTB mRNA before staining was designated as 1.0. The results are shown as the means ± SD of triplicate determinations.
as expected, the FRTL-5 and PC3 cells had been collected as TG positive, and TTF-1 positive and TG negative fractions, respectively. The three cell lines were then mixed and sorted into two fractions: the TG positive fraction corresponding to FRTL-5 cells and the TG negative and TTF-1 positive fraction corresponding to PC3 cells. 104 cells were collected in each fraction, and then the copy numbers of rat ACTB, rat TG, human ACTB, and human SFTBP mRNA in each fraction were measured (Table 1). In the TG positive fraction, rat ACTB and TG mRNA in all cases, but not human ACTB or SFTBP mRNA were detected. On the other hand, in the TTF-1 positive and TG negative fraction, human ACTB and SFTBP mRNA, but not rat ACTB or TG mRNA were detected, which suggested that,
4. Discussion In this study, we established a novel in-tube immunocytochemistry method that preserved cellular RNA even after staining, which made it possible to separate the stained cells by FACS. Thus, using this method any cells of interest can be separated by FACS using intracellular and/or nuclear antigens as markers, and then their biological characteristics can be analyzed by extracting their RNA and comparing their gene expression profiles.
Fig. 3. Flow cytometry and cell sorting. After immunocytochemistry, the FRTL-5, PC3, and 8305C cells were analyzed and sorted by flow cytometry with fluorescein-labeled TG antibody and R-PE labeled TTF-1 antibody. A: 8305C cells; B: FRTL-5 cells; C: PC3 cells; D: a mixture of 8305C, FRTL-5, and PC3 cells. (a) shows the gating range of the TG positive fraction (right quadrant), and (b) shows that of the TTF-1 positive and TG negative fraction (upper left quadrant).
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Table 1 Quantitative RT-PCR analysis of the cells before and after sorting. mRNA copy 104
TG positive cell
TTF-1 positive and TG negative cells
8305 cells before sorting
FRTL-5 cells before sorting
PC3 cells before sorting
Rat ACTB mRNA Human ACTB mRNA Rat TG mRNA Human SFTBP mRNA Rat TG/rat ACTB mRNA Human SFTBP/human ACTB mRNA
3.6 ± 0.1 N.D. 174.4 ± 31.1 N.D. 47.6 ± 8.1 —
N.D. 13.0 ± 2.0 N.D. 14.6 ± 1.8 — 1.01 ± 0.2
N.D. 3.6 ± 0.8 N.D. N.D. — —
6.0 ± 0.8 N.D. 166.6 ± 25.1 N.D. 28.2 ± 7.9 —
N.D. 9.0 ± 0.5 N.D. 11.6 ± 3.8 — 1.31 ± 0.5
104 stained cells were collected before and after sorting, and then their copy numbers of rat ACTB, ratTG, human ACTB, and SFTBP mRNAs were measured. The results are shown as the means ± SD of triplicate determinations. N.D., not detected.
Several studies have reported on the staining of intracellular antigens of fixed cells in suspension and the subsequent detection of the stained cells by FACS [13–16]. However, these studies did not confirm whether quantitative measurement of mRNA can be carried out after staining. When we started this project, the number of cells was greatly decreased after the staining procedure since fixed cells are sticky and so adhere to the plastic tube. We found that adding 0.1% Tween 20 to all buffers markedly increased the cell number after each washing step (data not shown). We also found that methanol supplemented with PEG was a suitable fixative for preserving RNA and cell morphology during staining, and we treated all media except those containing antibodies with DEPC as we found that use of the blocking reagent without DEPC treatment resulted in cellular RNA degradation (data not shown). Cells fixed in this manner can be stored at 80 °C for at least 2 weeks without changes in their cell morphology or the copy number of cellular RNA occurring. TG is one of the most abundantly expressed genes in differentiated thyroid cells [20,21], and TTF-1 is expressed in lung and thyroid cells [21,22]. We used FRTL-5 cells, a rat thyroid cell line, as TG and TTF-1 positive cells and PC3 cells, a human lung carcinoma cell line, as TTF-1 positive and TG negative cells. 8305C cells, which are derived from an undifferentiated thyroid carcinoma, do not express TG or TTF-1. The results regarding the gene expression levels of the cells in the TG positive fraction and the TTF-1 positive and TG negative fraction were quite reasonable. Rat ACTB and rat TG mRNA were detected in the TG positive fraction, which mainly contained FRTL-5 cells but did not containing 8305C or PC3 cells. Also, human ACTB and SFTBP mRNAs were detected in the TTF-1 positive and TG negative fraction, which mainly contained PC3 cells but did not containing FRTL-5 or 8305C cells. These results indicated that the gene expression profiles of cell fractions sorted by FACS are reliable and can be used to identify the biological characteristics of the targeted cells. 5. Conclusions We succeeded in establishing a novel immunocytochemistry method for mRNA quantification after FACS. These protocols will hopefully contribute to the accumulation of knowledge regarding human stem cells and CSCs, the biological characteristics of which are mostly unknown. Acknowledgments This research was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan via a Grant-in-Aid for Scientific Research C, 2008–2010, No. 20590570; a Research Grant from the Princess Takamatsu Cancer Research Fund 04-23606; and the Japanese Society of Laboratory Medicine Fund for the Promotion of Scientific Research.
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