interpretation of agilent 2100 bioanalyzer data. distinct 28s ribosomal subunit (or prok. 23s):...
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Interpretation of Agilent 2100 Bioanalyzer Data
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Distinct 28S ribosomal subunit (or prok. 23S): ideally 2X size of 18S
Distinct 18S ribosomal subunit (or prok. 16S)
No well defined peaks between ribosomal peaks
Flat baseline throughout electropherogram
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.
Marker
28S
18S
Intact Total RNA
~100 bp
Partially Digested Total RNA
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The 28S subunit often degrades firstDecrease in ribosomal peak intensities
Increase in intensity of smaller digested RNA fragments
Baseline between and to the left of ribosomal peaks becomes jagged
Total RNA with images like this are borderline. Re-extraction should be seriously considered.
Partially Digested Total RNA Using Trizol Extraction
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Combination of 5S, 5.8S, tRNAs, and an increase in digested RNAs
Decrease in ribosomal peak intensities
marker Digested RNA
~100 bp
Heavily Digested RNA
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Peaks shift to left
High digested RNA peaks
Samples of this quality, if labeled and hybed to a chip, might be in question.
~100 bp
Heavily Digested RNA Using a Hot Phenol with Beads Extraction
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Completely Digested RNA
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~100 bp
Marker
Characteristics of Good Labeled cRNA
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Marker
Small initial hump
Round Curve with a few small peaks
Smear
~1 kb
Labeling with Partial Fragmentation
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~1 kb
More defined initial hump
Labeled cRNA with this image are OK to fragment and hyb, but not without risk.
Insufficient Transcription During Labeling
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Peaks still present, but digested
These samples have failed, and are not ready for hybridization.
Properly Fragmented cRNA
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Fragmented samples with this image are ready for hybridization.
Marker
~100 bp
Low Quantities of Fragmented cRNA
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Peak heights are related to quantity. The lower the peak, the less fragmented cRNA is present.
Notice that the gel image looks normal.
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.
~100 bp
A Proper Ladder
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25 bp
200 bp
500 bp
~6 kb
4 kp1 kb
2 kb
Ladder That Has Not Been Denatured Sufficiently
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Degraded LadderFl
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Mechanical Spikes
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Mechanical spike
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 thequality of the RNA, labeled cRNA, fragmented cRNA, etc. and are OK to continue processing.
Enhanced Image of Mechanical Spike
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Notice classical W shape.
Bands are sharp and do not fade like real peaks.
28S
18S
Spike
Genomic DNA Contamination
Additional Genomic DNA Peak
Genomic DNA skewing 28S peak
Picture from Agilent Troubleshooting manual
Nanochip ContaminationF
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Could also result from fingerprints on focusing lens or on backside of chip.Be careful not to touch top or bottom of chip.
Electrode Cartridge Contamination
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28S
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Dirty or wet electrode cartridge. Make sure electrode cartridge is clean and dry.
Wavy Baseline Late Migration