1. Interpretation of Agilent 2100 Bioanalyzer Data

2. Intact Total RNA
Distinct 28S ribosomal subunit (or prok. 23S): ideally 2X size of 18S
Distinct 18S ribosomal subunit (or prok. 16S)
28S
18S
No well defined peaks between ribosomal peaks
Marker
Flat baseline throughout electropherogram
~100 bp
Marker
Small peaks are sometimes present after the marker at 24 – 29 seconds. These are represented by 5S and 5.8S subunits, tRNAs, and small RNA fragments about 100bp. These are especially noted when using phenol and trizol extraction methods. They can be removed by treating total RNA through Qiagen columns which removes small RNAs.

3. Partially Digested Total RNA
Total RNA with images like this are borderline. Re-extraction should be seriously considered.
Decrease in ribosomal peak intensities
The 28S subunit often degrades first
Increase in intensity of
smaller digested RNA fragments
Baseline between and to the left
of ribosomal peaks becomes jagged

4. Partially Digested Total RNA Using Trizol Extraction
Decrease in ribosomal peak intensities
~100 bp
marker
Digested RNA
Combination of 5S, 5.8S, tRNAs, and an increase in digested RNAs

5. Heavily Digested RNA
Samples of this quality, if labeled and hybed to a chip, might be in question.
~100 bp
Peaks shift to left
High digested RNA peaks

6. Heavily Digested RNA Using a Hot Phenol with Beads Extraction

7. Completely Digested RNA
~100 bp
Marker

8. Characteristics of Good Labeled cRNA
~1 kb
Marker
Round Curve with a few small peaks
Smear
Small initial hump

9. Labeling with Partial Fragmentation
Labeled cRNA with this image are OK to fragment and hyb, but not without risk.
~1 kb
More defined initial hump

10. Insufficient Transcription During Labeling
These samples have failed, and are not ready for hybridization.
Peaks still present, but digested

11. Properly Fragmented cRNA
Fragmented samples with this image are ready for hybridization.
~100 bp
Marker

12. Low Quantities of Fragmented cRNA
This sample may still be OK to hyb, but frequent failures have been reported with
cRNA levels this low. It is recommended that the fragmentation be repeated.
Peak heights are related to quantity. The lower the peak, the less fragmented cRNA is present.
~100 bp
Notice that the gel image looks normal.

13. A Proper Ladder
200 bp
25 bp
2 kb
500 bp
1 kb
4 kp
~6 kb

14. Ladder That Has Not Been Denatured Sufficiently

15. Degraded Ladder

16. Mechanical Spikes
These spikes are due to microparticulates and microbubbles. Dust is a common cause for these.
Make sure to quickly load your nanochips and keep your area clean. These do NOT affect the
quality of the RNA, labeled cRNA, fragmented cRNA, etc. and are OK to continue processing.
Mechanical spike

17. Enhanced Image of Mechanical Spike
Bands are sharp and do not fade like real peaks.
Spike
28S
Notice classical W shape.
18S

18. Genomic DNA Contamination
Genomic DNA skewing 28S peak
Additional Genomic DNA Peak
Picture from Agilent Troubleshooting manual

19. Nanochip Contamination
Could also result from fingerprints on focusing lens or on backside of chip.
Be careful not to touch top or bottom of chip.

20. Electrode Cartridge Contamination
Late Migration
Wavy Baseline
Dirty or wet electrode cartridge. Make sure electrode cartridge is clean and dry.