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Supporting Information

On-chip separation and analysis of RNA and DNA from single cells

Hirofumi Shintaku,1,4 Hidekazu Nishikii,2 Lewis A. Marshall,3

Hidetoshi Kotera,4 and Juan G. Santiago1* 1Department of Mechanical Engineering, 2Divisions of Blood and Marrow

Transplantation, and 3Department of Chemical Engineering, Stanford University,

Stanford, California 94305, United States 4Department of Micro Engineering, Kyoto University, Kyoto 606-8501, Japan

* To whom correspondence should be addressed. E-mail: juan.santiago@stanford.edu

This document contains supplementary figures and information further describing our

ITP based extraction and quantification of RNA from single cells

S-1 Electrical Lysis of Single Cells On-chip electrical lysis was first demonstrated by MacClain et al.1 using an AC electric

field of 75 Hz and 900 Vcm-1 with a DC offset of 675 Vcm-1. There has been a wide

variety of on-chip electrical lysis. For example, Gao et al.2 and Munce et al.3

demonstrated electrical cell lysis with relatively lower DC electric fields using

assistance of a high pH buffer (pH = 9.2) and mechanical shear induced by cell trapping

micro-structures, respectively. These studies used a saline-based buffer having high

salt concentration (order 100 mM) to compensate the cell osmotic pressure. We here

provide ITP chemistry using sucrose to increase osmolarity while achieving a cell lysis

buffer compatible with ITP (see Fig. S-2). We observed our ITP chemistry preserved

cell viability for at least 3 h. However, we note that this chemistry may provide some

stress to the cells and may change expression level. In this initial Technical Note

publication, we present an initial description of our single cell assay and provide strong

evidence that the assay can capture the heterogeneity due to the cell cycle. In future

work, we hope to more closely explore the effect of the solution on the RNA, including

correlation between pH of solution and expression level.

We introduced a single cell from the W reservoir into the injection channel,

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where it is between the W reservoir and the cross, by applying a vacuum to the S

reservoir. The length of the injection channel is 7.38 mm. The length between the N

reservoir and the cross is 3.925 mm. Taking advantage of the relatively short length

between the N and the W reservoirs, we applied a bipolar voltage pulse between N and

W to give a high intense electric field. We electrically and selectively lysed the single

cell isolated in the injection channel within 10 ms. (See the multimedia SI for the high

speed observation of cell lysing process.) Figure S-1B shows the voltage sequence for

electrical lysis followed by ITP. We used a bipolar pulse with an individual pulse of

duration of each of the two 100 ms. The bipolar nature of the pulse helped minimize

movement of the cell during lysing, and this aided visualization of lysing. We then

immediately applied potential to initiate ITP and extract RNA from the lysed cell by

switching voltages VW and VN to DC voltages.

In our protocol, we briefly evaluated the viability of the single cell isolated in

the injection channel in bright field imaging. Single cells with a blurred outline were

rejected as dead cells. The rejected single cells comprised about 5% of the population,

and this fraction of dead cells was consistent with cell viability test using calcein

visualizations (see Fig. S-2). We intentionally analyzed five dead cells and confirmed

these exhibited either negligible or much smaller RNA amounts.

For the demonstration of Fig. 1D, we used cells dyed with calcein (Calcein

AM 8011, Biotium, Inc) to visualize membrane permeability (see also Fig. S-2). Both

cells initially showed fluorescence due to the calcein. Cells exposed to the pulsed

electric field (cell 1) quickly lost fluorescence in the subsequent frame, consistent with

disruption of the cell membrane. In contrast, cells located in a channel branch outside

the electric field (cell 2 between the junction and the S reservoir) maintained

fluorescence and remained intact. This selective lysing was important to avoid

contamination from non-targeted cells. We observed very repeatedly that our bipolar

voltage pulse lysed exclusively the single cell isolated in the injection channel, and the

cells left in the reservoir were intact through the assay. Given the geometry of the

reservoir, we estimate the electric field in the reservoir is about 0.02% of that in the

injection channel. This field strength is small enough to avoid the lysing the cells in

the reservoir. Such use of geometry to selectively lyse cells is well characterized and

accepted (e.g., see reference 4).

We performed highly temporally resolved imaging of electrical lysing of

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single cells with a high-speed camera (Phantom Micro-4M, Vision Research). These

images showed disruption of the cell membrane within 10 ms (see the multimedia SI for

a high frame rate movie). We estimated the electric field in the injection channel as

270 kV/m. From the characteristic 14 µm diameter of cells, we therefore estimated the potentials induced across the cell membrane were order ~3 V. This was high enough

compared to the typical ~1 V break down voltage of cell membranes.

S-2 Selection of PVP Concentration to Isolate RNA We observed two (fluorescent) nucleic acid regions in all single cell experiments. The

first was a high-mobility zone that always focused in ITP interface. We attributed this

to total cytoplasmic RNA. The second region was a roughly ellipsoidal body with

characteristic major and minor radii of roughly 8 and 10 µm (see Fig. 1F). The second

region focused in the ITP interface only for PVP concentrations of about 0.2% or less.

At higher PVP concentrations, the second region never focused in ITP interface. We

attributed the second region to cell nuclei. After a series of preliminary experiments,

we determined 0.4% PVP concentrations sufficiently and repeatedly arrested the motion

of the cell nuclei so that they never co-focused in the ITP interface. See the

multimedia SI for additional information, including visualizations of separations and

focusing at low and high PVP.

We confirmed our attribution of the two fluorescent regions as cell nucleus vs.

total cytoplasmic RNA by a series of experiments using Hoechst 33342 dye (B2261,

Sigma-Aldrich, which is selective to DNA vs. RNA) and using RNase (RNase A,

QIAGEN). First, experiments with Hoechst 33342 (and not RNase) revealed that the

ITP interface showed negligible fluorescence compared to the negative control, while

cell nuclei showed significant fluorescence intensity (c.f. Fig. S-3). This is, of course,

consistent that the cell nuclei always contained DNA and that DNA was not focused

into the ITP interface. Second, we show experiments conducted with RNase mixed

into the LE (see Fig. S-4). With RNase, the ITP-focused zone signal reduced to values

on the order of negative controls (signal-to-noise ratios, SNR, of order 0.4). These

observations lend confidence to the conclusion that our method focuses cytoplasmic

RNA with ITP, while leaving behind cell nuclei. Our identification of cell nuclei

versus RNA was also consistent with our observations of pre- and post-lysis cell

morphology, including using overlaid transmitted light and fluorescence visualizations.

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S-3 Development of An Experiment Calibration Curve We used a series of ITP experiments with spiked synthetic RNA to build a calibration

curve for absolute quantitation of RNA mass. Our calibration process was similar to

that used by Persat et al.5 We used conditions and solutions identical to those of cell

experiments, and spiked with known concentrations of an RNA ladder (0.5-10 Kb RNA

Ladder, Invitrogen). We added 2 µL of the sample solution in the W reservoir and injected it into the injection channel by applying vacuum to the S reservoir. We

injected all of the dispensed sample solution in the W reservoir into the microchannel,

and then removed the vacuum from the S reservoir and dispensed TE solution in the W

reservoir. By this method, we exchanged 12 nL of the solution in the injection channel

with the sample solution. We then inserted electrodes to the W, N and E reservoirs

and initiated the ITP process to focus RNA at the ITP interface. We quantified ITP

peak SNR, and constructed the calibration curve shown in Fig S-5A. The standard

deviation normalized by the mean for the data was about 12%. We attribute at least

part of this variation to errors in fluid handling including pipetting and vacuuming.

The solid line shows a linear regression with coefficient R2 = 0.98. We used this

calibration curve to relate integrated fluorescence signal and absolute RNA amount in

the range of 0 to 60 pg. Our calibration approach is based on the hypothesis that the

ITP dynamics is consistent regardless of the presence or absence of a nucleus in the

microchannel. We tested this hypothesis by observing very consistent velocities of the

ITP interface under the constant applied voltage, implying negligible effects on the

electric field and flow dynamics.

S-4 Image Processing of Cell Nuclei We analyzed images of the nucleus to identify their boundaries and integrate

fluorescence intensity in the nucleus. To do so, for each nucleus, we set the focal

plane to the highest intensity. We detected the nucleus as an area with fluorescence

intensities higher than a specified threshold. To automate the threshold setting, we set

the threshold value to Ibk+ σ, where Ibk is the mean intensity of the background

fluorescence within the channel and σ is the standard deviation of this background. The fluorescence image in Fig. S-6A shows an image of a cell nucleus and the boundary

determined by this threshold. Fig. S-6B shows a plot of the intensity along the X-X’ line.

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The threshold is shown in the latter plot as a straight horizontal line. The parameters

of the nucleus were obtained using the function of ‘regionprops’ in MATLAB. We

present these integrated intensity measurements of DNA in the nucleus as relative mass

values and not absolute quantitation of mass. Absolute quantification of DNA inside

nuclei is challenging for three reasons. First, the labeling efficiency associated with

transport into cell nucleus and quantum yield of the dye in the cell nucleus is difficult to

determine in an absolute sense. Second, obtaining a control value of absolute DNA

amount in the nucleus is difficult. This is unlike the case of free RNA focused in an

ITP zone (where we can use spiked RNA focused into an ITP zone as a control).

Third, we note that the integrated fluorescence intensity showed variation of about 15%

as a function of the location of the focal plane within the channel depth. We

hypothesize this variation is associated with the three-dimensional structure of cell

nucleus.

S-5 Correlation between Absolute Amount of Extracted RNA and Relative Amount of DNA We examined the correlation between the quantified amount of extracted RNA mass

and the integrated fluorescence signals of corresponding, individual cell nuclei as

shown in Fig. 2C. We obtained a correlation coefficient of 0.62 (p = 5.5x10-12 against

the null hypothesis of no correlation), indicating a significant positive correlation.

This correlation is itself interesting because RNA and DNA are known to correlate with

cell cycle and with each other (e.g., reference 6). We assume no functional model

relating DNA vs. RNA and chose this analysis over a standard regression analysis, as

we have no evidence or reason to introduce a specific mathematical expression relating

RNA and DNA amounts. Further, we note that we also obtained a significant and

positive correlation coefficient of 0.87 (p<0.001) using our FACS data.

We have various uncertainties due to a) optics, b) degree of completion of

dye/molecule binding reaction, c) variations in the ITP chemistry and process (including

pipetting errors), and d) extraction efficiency. From our calibration experiments, we

estimated that the combined effects of a)-c) result in a 12% of coefficient of variation.

This variation is much lower than coefficients of variation for the RNA and DNA

quantifications: 39% and 34%, respectively. The strong positive correlation

therefore suggests that the variation found in the amount of the extracted RNA was not

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due to variations in the extraction efficiency. Further these observations of positive

correlation and the relative variations in calibration experiments versus cell data each

support the conclusion that the observed variations in the single cell data can be

attributed to biological variations and not just experimental measurement uncertainty.

There is a possibility that the uncertainties of the RNA and DNA amounts could be

correlated (e.g., due to illumination fluctuation). However, we carefully chose an LED

excitation source rather than a mercury lamp to minimize fluctuation. We argue that

this effect is also included in variation source a) above. Further, RNA may exist in the

nucleus and affects the DNA measurement. The observed positive coefficient does not

reject this possibility, but it does support that the variation is due to cell heterogeneity.

This attribution to cell heterogeneity is also strongly supported by the agreement

between our data and the FACS data.

Figure S-7 shows two-dimensional Gaussian mixture analysis of the data

relating extracted RNA amount and the relative integrated fluorescence of the nucleus.

We performed this analysis using the function ‘gmdistribution.fit’ in MATLAB. We

examined one, two, three and four two-dimensional Gaussian distributions to fit the data.

We evaluated the fitting result on the basis of Akaike’s information criterion (AIC) and

Bayesian information criterion (BIC) values as listed in Table S-1. The minimum AIC

and BIC values for the two-dimensional Gaussian distribution suggest the two Gaussian

distributions provides a best fit without over-fitting of the data.

S-6 Protocol of FACS Analysis A20 cells were fixed by 70% ethanol at about 23oC for 16 h. Fixed cells were washed

once and stained for 45 min at 37oC with Hoechst 33342 (20 µg/ml, Sigma-Aldrich) in

HBSS plus 2% FBS. Pyronin Y (1 µg/ml, Sigma-Aldrich) was then added to the staining solution and the cells were incubated 15 min at 37oC. All samples were

analyzed by LSR-II-UV flow cytometer (BD Bioscience) at the Stanford Shared FACS

Facility.

References (1) McClain, M. A.; Culbertson, C. T.; Jacobson, S. C.; Allbritton, N. L.; Sims, C. E.;

Ramsey, J. M. Anal. Chem. 2003, 75, 5646-5655.

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(2) Gao, J.; Yin, X. F.; Fang, Z. L. Lab Chip 2004, 4, 47-52.

(3) Munce, N. R.; Li, J. Z.; Herman, P. R.; Lilge, L. Anal. Chem. 2004, 76, 4983-4989.

(4) Han, F.; Wang, Y.; Sims, C. E.; Bachman, M.; Chang, R.; Li, G. P.; Allbritton, N. L.

Anal. Chem. 2003, 75, 3688-3696.

(5) Persat, A.; Marshall, L. A.; Santiago, J. G. Anal. Chem. 2009, 81, 9507-9511.

(6) Crissman, H. A.; Darzynkiewicz, Z.; Tobey, R. A.; Steinkamp, J. A. Science 1985,

228, 1321-1324.

Table S-1 AIC and BIC-values corresponding to fits with one, two, three, and four

two-dimensional Gaussian distributions obtained from 100 single cells. The minimum

values for both the AIC and BIC at two two-dimensional Gaussian suggest the

two-Gaussian fit provides the best trade-off between goodness of fit and over-fitting.

Fitting with three and four Gaussians resulted in over-fitting.

Number of

Gaussians 1 2 3 4

AIC-values 527 494 499 505

BIC-values 540 523 543 565

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Figure S-1 (A) Microfluidic chip geometry. To measure the current during the ITP

process, we connected in series an input of a DAQ (NI USB-6009, National

Instruments) to the electrode placed in the S reservoir. We estimated the current by

dividing the voltage by the input resistance, 144 kΩ, of the DAQ. The input resistance was negligibly small compared to the microchannel resistance that was approximately

100 MΩ. (B) Voltage sequence for electrical lysis and ITP.

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Figure S-2 Bright field (top) and fluorescence microscopy images (bottom) of A20

cells suspended in the following three buffers: 50 mM Tris and 25 mM HEPES (left);

50 mM Tris, 25 mM HEPES and 175 mM sucrose (middle); the PBS (right). We dyed

the cells with calcein to examine the cell viability. The cells suspended in the buffer

with sucrose showed similar morphology with those in the PBS, indicating successful

compensation of the osmotic pressure.

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Figure S-3 Fluorescence signal of the ITP interface visualized with Hoechst 33342 of

the DNA specific dye. The signal with a single cell showed no significant difference

from that of the control (p = 0.57). The nucleus showed significantly high fluorescence

signal compared to the control (p = 0.0063). We concluded that the DNA extracted to

the ITP interface was negligible.

Figure S-4 Time-resolved images of the fluorescence of the ITP interface. The RNA

fluorescence from the experiment with a single cell was significantly higher than that

with the negative control. The fluorescence at the ITP interface approached that of no

cell for the cases where we added RNase to the LE, indicating the focused and

visualized molecules in the ITP interface were RNA.

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Figure S-5 (A) Calibration curve for the RNA quantification. We obtain the curve as

the relation between the fluorescence signal and the amount of RNA spiked into the TE

in the injection channel. (B) Extracted RNA amount from 100 single cells in

chronological order.

Figure S-6 Detection of a nucleus by the image processing. (A) Two dimensional

fluorescence image of a nucleus. (B) Spatial distribution of fluorescence intensity along

line X-X’ and threshold for dissection.

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Figure S-7 Two-dimensional Gaussian mixture analysis of the relation between the

extracted RNA amount and the fluorescence signal of nucleus. (A) Fitting with two

two-dimensional Gaussian distributions; and (B) fitting with three two-dimensional

Gaussian distributions.