Microfluidic analysis of DNA and RNA

From LabAutopedia

A LabAutopedia invited article

Microfluidic analysis of DNA and RNA
Derived from application notes from Caliper LifeSciences

Slab-gel electrophoresis is the historical standard method to determine the size, concentration, and purity of analytes such as DNA and RNA. Seeking to improve upon this methodology, much effort has been given to transitioning this analysis to the microfluidic, or lab-on-a-chip technology.^[1]^[2]^[3]^[4]^[5]^[6] Microfluidics refers to actively controling fluids in substrates with microfabricated channels of a few micrometers in dimension. Such systems utilize microfabrication techniquies developed for the semiconductor industry for the construction of three-dimensional networks of channels and components, and they provide a high level of control over the molecular structure of channel surfaces. One appeal of microfluidics is that the microchips require only a small amount of sample and reagent for each process—only a few tens or hundreds of nanoliters compared with the 100 ml or more required for macro-scale assays. Microfluidics technologies are easily automated to do routine assay and sample preparation on standardized chips with little human intervention.

When the dimensions of a device or system become small enough, particles suspended in a fluid become comparable in size to the device itself, which dramatically alters system and biochemical behavior. Microscale reactions may occur at different speeds from macroscale reactions because of the unique physics of small fluid volumes. Although fluid properties remain the same at the microscale, surface tension, viscosity, and electrical charges can become dominant forces on a fluid because the surface-to-volume ratio is much greater than for macroscale systems. Heat transfer and mass transfer function in the microscale domain are also quite different. At large scales, the inertia of the fluid is important, whereas at smaller scales it is not. Macroscale fluid transport systems usually employ mechanical pumps that exploit inertia for fluidic movement. Because fluidic inertia is negligible, microscale systems can employ other simpler fluidic transport approaches, such as applying voltages to fluids encased in a channel.

Microfluidic assay technology aims to take advantage of the unique characteristics of the micro-environment to integrate several sequential experimental steps into one process to obtain a complete “laboratory on a chip”. This leads to several advantages over the traditional methodologies:

LabChip bottom view - detection window and sipper cannula

Automated sampling from 96- and 384-well plates transfer plates from a thermal cycler directly into the microfluidic system, no sample prep or transfer needed.
More precise, accurate and reproducible data than gels.
Quantitative sizing and concentration data are automatically reported as each sample is analyzed.
Direct Generation of Digital Data – Eliminates the need for photo-documentation of gels.
Versatility – RNA, DNA, and protein analysis can be performed using the same system.
Higher Sample Throughput – Complete analysis for hundreds of samples in just a few hours; no further processing required.

System Specifications

Height 464 mm (18.28 in)
Width 472 mm (18.60 in)
Depth 649 mm (25.57 in)
Weight 45.5 kg (100 lbs)
Temperature Range 18-26 °C (64-79 °F)
Power Requirements 100/110/230 VAC, 50/60 Hz
Power Consumption 460 VA (460 W)
Plate Formats 96- and 384-well
Excitation/Emission Wavelengths 635 and 700 nm
Humidity Range 30-70% relative, non-condensing

Clockwise from top right: Full microfluidic analysis instrument; microfluidic chip; placement of microfluidic chip in extendable drawer, placement of sample-containing microplate onto drawer (below chip drawer)

Operation

Microfluidic electrophoresis is performed on a small, microfluidic chip. Prior to RNA or DNA analysis, reagents are loaded into the individual wells of the chip. These wells are connected to small plates of quartz etched with tiny microchannels about the size of a human hair. When the chip is loaded into the microfluidic system, its wells interface with platinum electrodes that provide voltage and current control. The system robot moves the microtiter plate wells directly under the chip’s capillary ‘sipper’, and approximately 150 nL of sample is aspirated onto the chip. The system uses high-voltage power supplies (currents ±50μA, ±1% and voltages 100–3000V ±2%) to drive the chip-based electrophoresis in the short channels to reduce the separation times to seconds per sample.

The basis of electroosmotic flow is the formation of an electrical double layer at the stationary/solution interface. silanol groups form the inner surface of the microchannel. These silanol groups are ionized above pH3. Thus, the inner surface of the channel is negatively charged (see video below). In solutions containing ions, the cations will migrate to the negatively charged wall. This forms an electrical double layer. When an electrical potential is applied to the column, with an anode at one end of the column and a cathode at another, the cations will migrate towards the cathode. Since these cations are solvated and clustered at the walls of the channel, they drag the rest of the solution with them, even the anions. This results in an electroosmotic flow, not to be confused with the electrophoretic migration.

Individual sample analytes are separated electrophoretically (video, below left) and the bands are detected via laser induced fluorescence (LIF). Sizing and concentration for each band are determined using both a ladder and internal markers. Because the sipper is rinsed between samples, cross-contamination or carryover is eliminated.

Size-based separations of protein or DNA samples can be achieved by loading specific channels of the chip with a sieving polymer solution. Rapid separations can be performed because the microfluidic channels dissipate heat with extreme efficiency – very high electric fields can be used with no measurable heating. The separation media in these assays contain fluorescent stains that bind to protein or DNA molecules non-covalently. Sample peaks are detected as they pass through a laser spot in the chip’s detection window.


Video of microscale electrophoretic separation	Animated depiction of ElectroOsmotic Flow

DNA analysis: Agarose (left) and comparable microfluidic (right).	RNA analysis. Visual representation of the analysis as a signal trace (left), and as a gel-like image (right).

Data processing and system control

Data acquisition and processing from a microfluidic device is a far more precise, accurate and reliable method of generating data than gel scanning and photo-documentation. System software automatically calculates the size and concentration of each protein or DNA fragment, evaluates the integrity of RNA, and provides digital results. Data are reported in real-time as each sample is processed. Results are displayed in a tabular format, an electropherogram view and in a gel-like image.