mRNA detection of individual cells with the single cell nanoprobe method compared with in situ hybridization
© Uehara et al; licensee BioMed Central Ltd. 2007
Received: 23 May 2007
Accepted: 10 October 2007
Published: 10 October 2007
The localization of specific mRNA generates cell polarity by controlling the translation sites of specific proteins. Although most of these events depend on differences in gene expression, no method is available to examine time dependent gene expression of individual living cells. In situ hybridization (ISH) is a powerful and useful method for detecting the localization of mRNAs, but it does not allow a time dependent analysis of mRNA expression in single living cells because the cells have to be fixed for mRNA detection. To overcome these issues, the extraction of biomolecules such as mRNAs, proteins, and lipids from living cells should be performed without severe damage to the cells. In previous studies, we have reported a single cell nanoprobe (SCN) method to examine gene expression of individual living cells using atomic force microscopy (AFM) without killing the cells.
In order to evaluate the SCN method, we compared the SCN method with in situ hybridization (ISH). First, we examined spatial β-actin mRNA expression in single living cells with the SCN method, and then the same cells were subjected to ISH for β-actin mRNA. In the SCN method, quantity of β-actin mRNA were analysed by quantitative PCR, and in ISH we used intensity of ISH as a parameter of concentration of β-actin mRNA. We showed that intensity of ISH is higher; quantity of β-actin mRNA detected by the SCN method increased more.
In this study, we compare the SCN method with the ISH. We examined β-actin mRNA expression in single cells using both methods. We picked up β-actin mRNA from several loci of a single living cell using an AFM nanoprobe, and identical cells were subjected to ISH. The results showed a good correlation between the SCN method and ISH. The SCN method is suitable and reliable to examine mRNAs at medium or higher expression level.
In situ hybridization (ISH) is a powerful molecular tool used to visualize nucleic acids, and it has attributed significantly to the advancement of the study of gene expression in cells and tissues. ISH was invented by two groups in 1969 [1, 2]. Around that time, only radioisotope (RI) was available to label nucleic acids. But nowadays, non-RI ISH can be preformed based on synthesis of nucleotides containing certain functional groups and synthesis of a modified oligonucleotide by Digoxigenin (DIG) system [3–6]. Its primary advantage over the Northern blot and reverse transcription polymerase chain reaction (RT-PCR) is its ability to detect localization of specific mRNA to a particular cell or a particular region in a cell. So ISH are applied for bacteria, culture cells, tissue section and whole mount embryo [7–11]. However, ISH cannot examine time-lapse change of identical cells because the cells have to be fixed.
We reported a single cell nanoprobe (SCN) method to examine mRNA expression without killing cells in a previous report [12–14]. In the method, an atomic force microscope (AFM) is used as a manipulator to obtain cell components containing mRNA from the target living cells. AFM has been applied for various biological samples because it can be operated in solution [15–19]. An AFM probe is inserted into the living cells to extract mRNAs. Obtained mRNAs are subjected to RT-PCR and then to nested PCR or quantitative PCR. Since the AFM has high positional and loading force control, extraction of cell components without severe damage to the cells is possible. By using the SCN method, we examined time-lapse mRNA expression change and mRNA localization in single living cells [12–14]. In those studies, we showed that the SCN method has the possibility of compensating for the disadvantages of ISH in the case of the single-cell study.
Results and discussion
The table summarizes the comparison between the average of IISH and the SCN method. In this table, the averages of IISH were generated from the range of 1.4 × 1.4 μm based on the position inserted by an AFM probe which was centered. We used the AFM probes with square pyramid shapes, whose height, horizontal length and 1/2 corn angles were 3, 4 μm, and 35°, respectively. So if we assume that the AFM probe is inserted into the cell by 1 μm, the range is 1.4 × 1.4 μm. In the table, when the average of IISH showed 0 to 0.1, β-actin mRNA was not detected by the SCN method. When the average of IISH was 0.1 to 0.25, β-actin mRNA was detected by the SCN method in low probability (33%) and low quantity. When the average of IISH was over 0.25, the probability of β-actin mRNA detection was 100%. However, the average quantities of β-actin mRNA detected by the SCN method were 50 and 120 molecules within the range of 0.25 to 0.4 and over 0.4, respectively. Based on this, as IISH increased more, the probability and quantity of β-actin mRNA detected by the SCN method increased more. These results indicated a proportional relation between the results of the ISH method and the SCN method.
Comparison between single cell nanoprobe method and ISH result (± S.D)
Single cell nanoprobe method
β-actin mRNA detection probability
0%(n = 5)
33%(n = 6)
100%(n = 7)
100%(n = 3)
Average number of detected β-actin mRNA
We showed the correlation between ISH and the SCN method. The SCN method can examine time-dependent mRNA expression of single living cells, but it is limited to the analysis of the fine localization of mRNA in the cells. ISH can examine mRNA expression of the whole cells with higher resolution, but time-lapse analysis cannot be done. Besides, the SCN method is suitable and reliable to examine mRNAs at medium or higher expression level. By using both methods, more accurate information about mRNA expression of single cells is available.
Preparation of cells
Rat fibroblast-like VNOf06 cells derived from the vomeronasal organ  were grown in 35 mm Petri dishes in Dulbecco's minimum essential medium (DMEM)/F12 supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (FBS). The cells were washed three times with DMEM/F12 without FBS and used for the AFM experiments.
The single cell nanoprobe (SCN) method
Briefly, the AFM probe (NP, Digital Instruments, Santa Barbara, CA) was positioned onto a target region of cells under the observation of an inverted phase-contrast microscope. The AFM probe was then inserted into the target cell using the step motor of the AFM (NVB-100, Olympus, Inc.), and held for about 30 s to allow the AFM probe to bind the cell components containing mRNA with physical adsorption. The AFM probe was lifted off the cell and placed into a PCR tube.
The reagents and primers of RT-PCR and quantitative PCR were used as previously described [12, 13]. RT-PCR was performed with a one-step RT-PCR kit (Qiagen, Valencia, CA). First-strand cDNA synthesis was performed at 50°C for 30 min, at which time the reaction was heated to 95°C for 15 min to activate HotStrTaq DNA polymerase. The amplification reaction was carried out for 30 cycles, and each cycle was 94°C for 45 s, 55°C for 45 s, and 72°C for 1 min, followed by a final 10 min elongation at 72°C. Quantitative PCR was performed with an Applied Biosystems Prism 7000 and the SYBR Green 1 PCR Mastermix (Qiagen, CA, USA) following previous studies [12, 13].
ISH for β-actin mRNA of single cells
Digoxigenin (DIG) labeled RNA probe preparation
β-actin cDNA [224–987 bp] was prepared by RT-PCR. β-actin cDNA was inserted into pGEM(R)-T Easy vector (promega), and subcloned. The direction of the inserted cDNA was examined by restriction enzyme and by its sequence. Antisense DIG-labeled RNA probe was prepared by SP6 and T7 RNA polymerase (stratagene) and 10 × DIG labeling mix (Roche). The efficiency of DIG labeling was examined by dot-blotting.
Cell preparation for ISH
After picking up mRNA by the SCN method, the cells were washed by PBS 3 times and fixed in 4% paraformaldehyde(PFA)/PBS for 30 min. From this point, all treatments were performed under RNase free condition. After PBS washing, the cells were treated by 1 μg/ml proteinase K (Invitrogen) for 5 min at 37°C, washed in PBS, refixed in 4% PFA/PBS for 10 min at RT, neutralized in 0.2% glycine/PBS for 2 min, 0.2 N HCl at RT for 20 min and washed with PBS two times.
Hybridization and detection by alkaline phosphatase reaction
Hybridization solution (60% formamide (deionized), 2 × SSC (1 × SSC is 150 mM NaCl, 15 mM), 10 mM EDTA, 25 mM NaH2PO4, 5% dextran sulfate and RNA probe (added before use)) was add to the cells described above and incubated overnight at 55°C. The RNA probe concentration was determined before the experiment and was adjusted to be 0.1 ng/μl. After overnight incubation, the cells were washed in the following order: 5 × SSC/50% formamide 30 min 50°C two times, TNE buffer 5 min (10 mM Tris, 0.5 M NaCl, 1 mM EDTA pH7.5), 20 g/ml RNase/TNE buffer 30 min 37°C, 2 × SSC 30 min 50°C two times, 0.2 × SSC 30 min 50°C two times, blocking solution (1% Blocking Reagent (Roche)/TBS (0.1 M Tris-HCl pH7.5, 0.15 M NaCl)) 30 min. The cells were then incubated in anti-DIG Fab fragment(Roche) diluted 1:500 with blocking solution for 60 min. After washing with TNT buffer (0.2% Tween20/TBS) 15 min two times and AP buffer (0.1 M Tris-HCl pH9.5, 0.1 M NaCl, 50 mM MgCl2), the cells were stained by DIG Nucleic Acid Detection kit (Roche) for 6 hours using alkaline phosphatase reaction of NBT/BCIP. After PBS washing, the cells were embedded in PermaFluor Mountant Medium (Thermo, USA) and obsreved by a phase-contrast microscope and a bright-field microscope.
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