Interpretation of Agilent 2100 Bioanalyzer Data

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Interpretation of Agilent 2100 Bioanalyzer Data
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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.
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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
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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
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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
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Heavily Digested RNA Using a Hot Phenol with
Beads Extraction
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Completely Digested RNA
100 bp
Marker
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Characteristics of Good Labeled cRNA
1 kb
Marker
Round Curve with a few small peaks
Smear
Small initial hump
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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
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Insufficient Transcription During Labeling
These samples have failed, and are not ready for
hybridization.
Peaks still present, but digested
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Properly Fragmented cRNA
Fragmented samples with this image are ready for
hybridization.
100 bp
Marker
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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.
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A Proper Ladder
200 bp
25 bp
2 kb
500 bp
4 kp
1 kb
6 kb
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Ladder That Has Not Been Denatured Sufficiently
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Degraded Ladder
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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
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Enhanced Image of Mechanical Spike
Bands are sharp and do not fade like real peaks.
Spike
28S
Notice classical W shape.
18S
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Genomic DNA Contamination
Genomic DNA skewing 28S peak
Additional Genomic DNA Peak
Picture from Agilent Troubleshooting manual
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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.
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Electrode Cartridge Contamination
Late Migration
Wavy Baseline
Dirty or wet electrode cartridge. Make sure
electrode cartridge is clean and dry.