Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization

Abstract

We have developed a novel method for genetic characterization of single cells by integrating microfluidic stretching of chromosomal DNA and fiber fluorescence in situ hybridization (FISH). In this method, individually isolated cell nuclei were immobilized in a microchannel. Chromosomal DNA was released from the nuclei and stretched by a pressure-driven flow. We analyzed and optimized flow conditions to generate a millimeter-long band of stretched DNA from each nucleus. Telomere fiber FISH was successfully performed on the stretched chromosomal DNA. Individual telomere fiber FISH signals from single cells could be resolved and their lengths measured, demonstrating the ability of the method to quantify genetic features at the level of single cells.

Appendix

The shear stress distribution for Poiseuille flow in a rectangular channel can be expressed in terms of the deviation from that in an infinitely-wide channel (plane Poiseuille flow, Boussinesq 1868, White 1974). The wall shear stress in plane Poiseuille flow is expressed as

$$ \tau_{{wall}}^{\infty } = \frac{{6\mu v_{{av}}^{\infty }}}{h} = \frac{{6\mu {v_{{av}}}}}{{h(1 - \delta )}}, $$

where

$$ \delta = \frac{{192}}{{{\pi^5}A}}\mathop{\sum }\limits_{{n = 1,3,5, \ldots }}^{\infty } \frac{{\cosh \left( {n\pi A} \right) - 1}}{{{n^5}{ \sinh }(n\pi A)}}. $$

${\text{At }}\mu = {1} \times {1}{0^{{-{3}}}}{\text{Pa}} \cdot {\text{s}}$, h = 250 μm, and channel aspect ratio A = w/h = 4, δ and $\tau_{{wall}}^{\infty }$ are calculated to be 0.158 and 6.33 dynes/cm² respectively.

For a channel of width w and height h, the shear stresses normalized by $\tau_{{wall}}^{\infty }$ in the channel are given by

$$ \begin{gathered} \tau = \left( {1 - 2z} \right) - \frac{8}{{{\pi^2}}}\mathop{\sum }\limits_{{n = 1,3,5, \ldots }}^{\infty } \frac{{\sinh \left( {n\pi y} \right) + { \sinh }(n\pi \left( {A - y} \right))}}{{{n^2}{ \sinh }(n\pi A)}}{ \cos }(n\pi z), \hfill \\ \quad \quad \quad \quad \quad \quad \quad 0 < y < A = \frac{w}{h}\quad \quad \quad 0 < z < 1 \hfill \\ \end{gathered} $$

where the coordinates along the width and height, y and z, respectively, have been normalized by the channel height h and channel aspect ratio A.