Single-cell study of the extracellular matrix effect on cell growth by in situ imaging of gene expression

The effect of extracellular matrix stiffness on cell growth and the underlying molecular mechanism was investigated using an in situ single-cell imaging of gene expression method based on rolling circle amplification.

11. Figure. S9. Quantitative analysis of mRNAs GAPDH by RT-qPCR. S9 12. on the slide. A blocking solution containing 10 ng/μL sonicated salmon sperm DNA, 2 × SSC buffer and 0.05% Tween-20 was added and the slide was incubated for 20 min at 37 °C. Washing was performed with 2× SSC buffer once. For hybridization of the RCA amplicons, 3 μL of hybridization mixture containing 100 nM fluorophore-labeled detection probes and 2 × SSC buffer was added and incubated at 37 °C for 30 min. After hybridization, the slides were mounted with Fluoromount-G and were ready for imaging.
SEM and FTIR Spectroscopy. Hydrogel samples on coverslips were swelled in PBS overnight, then flash-frozen by plunging into liquid nitrogen. The substrates were freeze dried overnight. Then, the gels on coverslips were sputter coated with platinum. Gel surface topography was viewed in a SU-8010 scanning electron microscope (HITACHI, Japan). Attenuated total reflection Fourier Transform IR spectra (ATR-FTIR) were carried out on an UATR Two FT-IR spectrometer (Perkin Elmer, USA). Briefly, the freeze-drying hydrogel samples were put on the window and the spectra was scanned from 400 cm -1 to 4000 cm -1 .
Image acquisition and analysis. The fluorescence images of RCA amplicons were acquired using a Leica TCS SP5 inverted confocal microscope (Leica, Germany) with a 63 × oil-immersion objective. For Alexa488-labeled and Cy5-labeled probe, Ar+ (488 nm) laser and HeNe633 (633 nm) laser were used as the excitation source, and detected with a 500-535 nm bandpass filter and a 650-750 nm bandpass filter, respectively. To ensure that all RCA amplicons were imaged, images were collected as z-stacks. Stacks of images were taken with 0.15 μm between the z-slices for imaging amplicons in vitro and 0.2 μm between the z-slices for imaging in situ in cells, and combined to a single image by MIP using Image J version 1.46r software. The superbright spot supposed to be a single amplicon was distinguished from the background signal by setting the intensity threshold with Image J version 1.46r software. To determine the copy number per cell, the superbright spots were firstly marked with a pseudo color in the fluorescence images and the outline of the cell was marked out from the bright field images. The number of mRNA was determined by counting the isolated amplicon signals inside the outline of the cell.

Real-time quantitative PCR (RT-qPCR) analysis of mRNA inside cells.
The cell lines MCF-7 were harvested after counting of cells and total RNA was extracted using TransZol following the manufacturer's instructions. The total RNA concentration and quality were investigated on a NanoDrop spectrometer (ND_200, NanoDrop Technologies, USA). The cDNA samples were prepared using TransScript one-step gDNA removel and cDNA synthesis. Briefly, a total volume of 20 μL solution containing 2 μL of the total RNA (50 ng-5 μg), 1 μL anchored oligo(dT)18 primer (0.5 μg/μl), 10 μL 2 × TS reaction mix, 1 μL TransScript RT/RI enzyme mix, 1 μL gDNA remover and 5 μL RNase-free water was incubated at 42 °C for 15 min followed by heat inactivation of reverse transcriptase for 5 s at 85 °C. The cDNA samples were stored at -80 °C for future use.
qPCR analysis of mRNA was performed with SYBR select master mix according to the manufacturer's instructions on a Bio-Rad C1000TM (Bio-Rad, USA). Templates for standard curves for the different genes were created by PCR. For this PCR, the 20 μL reaction solution contained 2 μL of cDNA sample, 10 μL 2 × SYBR Select master mix, 2 μL forward primer (5 μM), 2 μL reverse primer (5 μM), 4 μL RNase-free water. The total PCR volume was 20 µl and the PCR was carried out with 2 min at 95 °C, followed by cycling 45 × (95 °C for 15 s and 60 °C for 1 min), and finished with 60 °C for 5 min. Ct values were converted into absolute GAPDH copy numbers using a standard curve from a control RNA (human GAPDH mRNA in RevertAid First Strand cDNA Synthesis Kit). A standard curve was prepared from cDNA solutions corresponding to the serially diluted solutions of human GAPDH mRNA. The volumes and components of reverse transcription and qPCR reaction mixtures were the same as those for the test samples. Obtained results are presented as the copies of mRNA per cell. The experiment was repeated three times. The copy number of target mRNAs ACTB, PFN1, and CFL1 was evaluated by referring to the expression of GAPDH mRNA using the 2 -ΔΔCt method. [1] Calculations of transcript copy numbers were based on the number of counted cells at harvest. To demonstrate the feasibility of rolling circle amplification for RNA detection, we first did in vitro experiment to characterize the sensitivity and selectivity of rolling circle amplification. The amplification signal was obtained by fluorescence spectral. A series of contrast experiments was carried out, the data were shown in Figure. S1A. When the target sequence and padlock probe were presented, there generated strong fluorescence (red), while only the padlock probe involved, just faint fluorescence was produced (pink), indicating that the RCA reaction results in strong fluorescence signal amplification and low background fluorescence (shown in Figure S1B). Besides, we designed a random sequence to replace the padlock probe, added to the system contained target sequence, there generated weak fluorescence (purple), meaning that padlock probe can recognize the target sequence specifically. Further, a blocked sequence perfectly binding with the target sequence was added to the system contained target indicating that a large number of RCA products with extremely large molecular weight were produced by RCA, as shown in Figure S1C. According to the results of the fluorescence spectra and gel electrophoresis characterization, the padlock probe can specifically recognize and effectively amplify the target sequence by RCA. The ability of this approach for quantitative detection of target DNA in vitro was investigated and the result is shown in Figure S2.  To further confirm the ability for quantitative detection of real mRNA by RCA, GAPDH mRNA was used as target mRNA and the RCA products (RCPs) were labeled with a 22-nt fluorescent complementary oligos, resulting in bright dots when dipped into a coverslip. We acquired the RCPs by RCA using different concentrations GAPDH mRNA, respectively and then dipped into the surface of a coverslip. A series of fluorescence images of the RCPs was captured by confocal microscopy and the results were shown in Figure S3A. The RCPs conjugated with the fluorescent probe presented bright dots distributed uniformly in each image frame and these bright dots owned a similar fluorescent intensity ( Figure S3B). The average diameter of the bright dots measured from the fluorescence images was approximately 1 μm. RCPs could be easily detected and identified by a conventional fluorescence microscope, making it suitable for single molecule detection. The numbers of the bright dots per image frame were counted from the images and the relationship with the GAPDH mRNA concentration was shown in Figure S3C, indicating that the numbers of the bright dots per image frame increased linearly with the mRNA concentrations. A good linearity was obtained from 0.1 fM to 50 fM of GAPDH mRNA. previously published smFISH thresholding method. [2] The superbright spot supposed to be a single RCA amplicon was distinguished from the background signal by thresholding the images using Image J software. The threshold value was chose based on the smFISH thresholding method, which lied in a region of plateau over which the number of spots detected is insensitive to the threshold chosen. The outline of the cell was marked out from the bright field images. The number of RCA amplicons in single cells was determined by counting the isolated fluorescence spots inside the outline of the cell using ImageJ software. 11.08 ± 0.85 [a] This table presents the relative concentrations of acrylamide and bis-acrylamide and their elastic modulus and mass-swelling ratio (Qm) after polymerization in PBS. [3] [b] The mass-swelling ratio (Qm) is typically defined as the ratio of wet weight(Mw)to dry weight (Md)，at least 3 times per sample were statisticsed for these measurements.