BioMicroCenter:BioAnalyzer INFO

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The Agilent BioAnalyzer 2100 uses nanofluidics to provide quantitative information about biological samples using very small amounts of material.

DNA

The BioAnalyzer analyzes DNA using the same principles as slab agarose gels. A very small tube of gel is created and a fraction of the DNA loaded, along with a low and high size marker, and are placed at one end of the chip. Then, using a voltage gradient, the sample is run down the length of the chip. As the DNA passes a laser, it is detected. Subsequent samples are run down the same lane of the chip. Quantification is preformed relative to the sizing standards. Only 1ul of sample is required.

DNA - How it works (Agilent)

There are four different options for DNA arrays:

• DNA 1000 Kit: 25bp - 1,000bp, 0.1-50ng/uL
• DNA 7500 Kit: 100bp - 7,500bp, 0.1-50ng/uL
• DNA 12000 Kit: 100bp - 12,000bp, 0.1-50ng/ul
• High Sensitivity DNA kit: 50bp - 7,000bp, 5-500pg/ul

The BioMicro Center keeps both the DNA 1000 and High Sensitivity DNA kits in stock, if you think you require a different kit please let us know in advance.

RNA

The BioAnalyzer analyzes RNA using the same principles as denaturing slab agarose or polyacrylamide gels. A very small tube of gel is created and a fraction of the RNA loaded, along with a low marker, is placed at one end of the chip. Then, using a voltage gradient, the sample is run down the length of the chip. As the RNA passes a laser, it is detected. Subsequent samples are run down the same lane of the chip. Quantification is preformed relative to the sizing standards.

RNA - Analysis Specifications (Agilent)

RNA arrays are segregated by the concentration of RNA used and the type of RNAs being analyzed

• RNA Nano: 25-500 ng/ul total RNA
• RNA Pico: 0.05-5 ng/ul total RNA
• small RNA: 0.05-2 ng/ul miRNAs

RNA Integrity Number Guide

• Overview of Agilent software:

The software on the Agilent BioAnalyzer 2100 automatically calculates an RNA integrity number to an eukaryote total RNA sample. The sample integrity is no longer determined by the ratio of the ribosomal bands, but by the entire electrophoretic trace of the RNA sample. RINs are calculated whether there is a presence or absence of degradation of a given sample. RIN is independent of sample concentration, however is based on characteristics of several regions of the electropherogram (signal areas, intensities, ratios).

Degradation

• During degradation, the 28S band disappears faster than the 18s band, so with this, we can spot the beginning stages of degradation.
• The fast region: unexpected signal in fast region refers to how fact the degradation in the samples proceeded.
• Decrease in signal intensities for the ribosomal bands (28, 18s).
• Increase in smaller/ shorter fragments.
• Elevated baseline between the ribosomal peaks and also the lower marker.

RIN Upsets

If the software fines an unexpected peak or signal in certain critical regions, it will assign a RIN to a particular sample that has these anomalies.

• Critical regions: 5S region, fast region> not computed
• Non-critical: pre-regions, precursor-regions, post-region> RIN computed but will be low
• RIN can still be computed by increasing anomaly threshold settings (Set Pint Explorer)on the specific parameters reported on the errors page.
• Anomalies: Genomic DNA contamination, ghost peaks, spikes, wavy baselines

PROTEIN

The same principles for RNA and DNA apply for protein. Here, the model is SDS-PAGE with samples denatured before loading and run in reducing (+DTT) or non-reducing conditions. The standard and high sensitivity assays are moderately different from each other. Standard assays work like DNA and RNA in that the sample is added to a dye and run together iwn the lane. With the high-sensitivity system, the buffers are exchanged by stnadard chromatography to tune the pH and remove contaminanting chemicals before being labeled. the labeled proteins then are run on the chip - or can be used for additional purification steps before running. BMC is currently optomizing the High sensitivity assay.

Information from Agilent: Protein - How it works (Agilent)

   Protein arrays    Protein Weight    Sensitivity
   * Protein 80         5kD- 80kD          60ng/ul
   * Protein 230       14kD-230kD          30ng/ul
   * High Sensitivity  10kD-250kD         0.3ng/ul

FLOW CYTOMETRY

In addition to quantification of Nucleic Acid, the BioAnalyzer is capable of preforming simple two color flow cytometry assays. In brief, six samples each 10 µl with 20,000 prestained cells are loaded onto the chip and the fluorescence intensities in two channels for about 750 single cells per sample are measured within 25 minutes.

Information from Agilent: Cells - How it works (Agilent)