Bioanalyzer

The Agilent Bioanalyzer is used for RNA and DNA analysis sizing and quality analysis.

High sensitivity DNA chip

  • Primarily for used for determining quality of library preps for Illumina sequencing.  To select this option for ordering pick 'Illumina prep DNA' for nucleic acid type.

Total RNA quality (Nano chip and Pico Chip)

  • Software gives a RIN number to give a quality assessment of the total RNA. 
  • A specific assay is needed to determine RIN for eukaryotic, prokaryotic, or plant samples. Pick correct assay type during sample submission to let us know which to run.
  • The software determines the ratio of 18S and 28S ribosomal peaks, or 16S and 23S peaks for prokaryotes. The 28S/18S ratio should be about 2, but may be less with the RNA still being of good quality. 
  • More important than the ratio is the degradation of the ribosomal peaks. Look for the amount of degradation before and between the ribosomal peaks.
  • Plant assay compensates for the extra ribosomal peaks from chloroplasts before the 18S peak when determining RIN.

mRNA quality (Nano chip and Pico Chip)

  • Shows contamination with ribosomal RNA and the approximate the size range of the mRNA. No RIN number.

Quantification

  • The bioanalyzer also quantifies RNA or DNA, but other fluorescent based methods are more accurate.

Other Chips

  • Other chips are available for use on the same instrument on a custom basis. You can find more information on the web site at Agilent.

Sample Dropoff

  • Submit samples online. Information below on ordering chip type and assay.
  • Make sure sample names or numbers on tubes match the order names or sample ID numbers.
  • Supply samples in clearly marked 0.2 or 0.5 ml tubes
  • Bring your samples to ALS 3137C. 
  • Put your samples in one of our submission boxes with a slip of paper containing your name, your PI's name, date and Order ID#. 
  • Samples go in our -20°C freezer by the door in one of the boxes marked Bioanalyzer. 
  • For timely processing, let us know your samples have arrived.
  • Samples run by a lab technician as time permits.  May be a day or two before results are available

Sample Information for Submission

  • 1µl of RNA or DNA is loaded on the bioanalyzer.
  • It is best to quantify RNA before running on the bioanalyzer, and run all samples with the same concentration to make a good sample-to-sample comparison.
  • Overloading or underloading will give inaccurate concentration results, or may clog the channels.
  • Lower concentrations may not work as well depending on the broadness of the peaks and number of bands and how accurately the concentration was determined.
  • RNA samples are heat denatured before loading.
  • The Pico chip is much more sensitive to contaminants than the Nano chip.
  • Avoid salts and particulate by using dust free gloves and filter free tips if possible when preparing samples.

Select Chip Type

Chip type Sizing Range Quantitative Range Maximum Salt Concentrations Samples
per chip		 Minimum Sample Volume
RNA Nano  
Total RNA: 25–500 ng/μl
mRNA 25–250 ng/μl
 100 mM Tris
0.1 mM EDTA
or 125 mM NaCI
15 mM MgCI2		 12 2-3 µl
RNA Pico   Total RNA: 50–5000 pg/μl  (in water)*
mRNA 250–5000 pg/μl   (in water)*		 50 mM Tris
0.1 mM EDTA
or 125 mM NaCI
15 mM MgCI2		 11 2-3 µl
HS-DNA  50-7000 bp 5 - 500 pg/µl  10 mM Tris
1 mM EDTA		 11 2-3 µl

     *  Qualitative range

Select Assay

To select the correct assay for your sample type, see chart below. Consult with Katie Carter if you have questions about selection.

Nucleic Acid Type Organism Type Description/Defining Factor Assay
Total RNA    Eukaryotic  Only 18S, 28S bands, non-plant Eukaryotic Total RNA Assay
Extra Ribosomal Bands, ie Plants Plant RNA Assay
Prokaryotic  Extra Ribosomal Bands Plant RNA Assay
Just 16S, 23S bands Prokaryotic Total RNA Assay
       
mRNA Eukaryotic or 
Prokaryotic		 Determine ribosomal contamination mRNA Assay
Visualize only, shorter products <4000 Eukaryotic/Prokaryotic/Plant RNA assay
       
 cDNA or aRNA
  		 Eukaryotic or 
Prokaryotic 		 Illumina library prep High Sensitivity DNA Assay
Longer fragments >6000 for qPCR, use region feature in software mRNA Assay
Shorter fragments <4000 for qPCR Eukaryotic Total RNA Assay
       
Fragmented RNA
 		 Eukaryotic or 
Prokaryotic  		 Just visualize size Eukaryotic Total RNA Assay
Extract information from a region mRNA Assay
       
DNA seq prep for HiSeq Eukaryotic or 
Prokaryotic		 cDNA, amplicon, shotgun library, double stranded DNA 50-7000bp High Sensitivity DNA Assay

Data is available for downloading online after samples have been run.

Bioanalyzer Data Interpretation

  • Each lane contains a marker along with your sample. The marker is used to align the samples. The concentration and sizing is determined from the standard ladder loaded in lane one.
     
  • The 28S/18S ratio may be indicative of problems with the RNA, but not always. Even though the ideal ratio is 2.0, the bioanalyzer rarely reflects this ratio. If the ratio is greater than 2.0 it may indicate the presence of sheared single stranded genomic DNA which can run around the 28S band. If the ratio is less than 1.0, there are usually definite degradation problems with the RNA. More important than an absolute number is the visual sign of degradation.
     
  • The Agilent Expert software assigns a RIN number to each trace. It assigns a number according to how much signal is found between the 5S and 18S band, between the 18S and 28S bands, and after the 28S band. An RIN number of 10 is perfect score or excellent RNA. The software does not always call RIN numbers for prokaryotic RNA and the RIN can be misleading for samples containing additional RNA bands such as those from chloroplasts or a symbiotic RNA.

Here are some Agilent articles about interpreting the RIN number

  • RNA Integrity Number (RIN) - Standardization of RNA Quality Control [PDF]
  • RIN Users Guide [PDF]

See some examples of Bioanalyzer traces [PDF] to help with data interpretation

Free software from Agilent is available to view your data (.xad file) on a PC.

The bioanalyzer is run as a service by Katie Carter.

Cite this (2100 Bioanalyzer Instrument, RRID:SCR_018043)