|How does Agilent's 2100 Bioanalyzer work?
The chip accommodates sample wells, gel wells and a well for an external standard
(ladder). Micro-channels are fabricated in glass to create interconnected networks
among these wells. During chip preparation, the micro-channels are filled with a
sieving polymer and fluorescence dye. Then, twelve samples (NanoChip kit), or
eleven samples(PicoChip kit) and ladder with marker are loaded in each well.
Once the wells and channels are filled, the chip becomes an integrated electrical
circuit. The 16-pin electrodes of the cartridge are arranged so that they fit into the
wells of the chip. Each electrode is connected to an independent power supply that
provides maximum control and flexibility.
Charged biomolecules like DNA or RNA are electrophoretically driven by a voltage
gradient, similar to slab gel electrophoresis. Because of a constant mass-to-charge
ratio and the presence of a sieving polymer matrix, the molecules are separated by
size. Smaller fragments are migrating faster than larger ones. Dye molecules
intercalate into RNA strands and these complexes are detected by laser-induced
Data is translated into gel-like images (bands) and electropherograms (peaks).
An RNA 6000 ladder standard is run on every chip used as a reference for data
analysis. (either the RNA 6000 Nano- or Pico Ladder). The RNA 6000 ladder contains
six RNA fragments ranging in size from 0.2 to 6 kb (0.2 kb, 0.5 kb, 1.0 kb, 2.0 kb, 4.0
kb, and 6.0 kb) at a total concentration of 150 ng/µl (the Pico ladder at a total
concentration of 1 ng/µl).
During the chip run, the dye intercalates directly with the RNA and all bands pass the
detector at different speeds. An extra “lower” marker fragment is run with each of the
samples to compensate for drift effects that may occur during the course of a chip run.
The software automatically compares the unknown samples to the ladder fragments
to determine the concentration of the unknown samples and to identify the ribosomal
Next: How does intact total RNA looks like?
|RNA QUALITY CONTROL
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