Interpretation of agilent 2100 bioanalyzer data
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Interpretation of Agilent 2100 Bioanalyzer Data. 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.

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Interpretation of Agilent 2100 Bioanalyzer Data

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Interpretation of agilent 2100 bioanalyzer data

Interpretation of Agilent 2100 Bioanalyzer Data


Intact total rna

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.


Partially digested total rna

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


Partially digested total rna using trizol extraction

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


Heavily digested rna

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


Heavily digested rna using a hot phenol with beads extraction

Heavily Digested RNA Using a Hot Phenol with Beads Extraction


Completely digested rna

Completely Digested RNA

~100 bp

Marker


Characteristics of good labeled crna

Characteristics of Good Labeled cRNA

~1 kb

Marker

Round Curve with a few small peaks

Smear

Small initial hump


Labeling with partial fragmentation

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


Insufficient transcription during labeling

Insufficient Transcription During Labeling

These samples have failed, and are not ready for hybridization.

Peaks still present, but digested


Properly fragmented crna

Properly Fragmented cRNA

Fragmented samples with this image are ready for hybridization.

~100 bp

Marker


Low quantities of fragmented crna

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.


A proper ladder

A Proper Ladder

200 bp

25 bp

2 kb

500 bp

1 kb

4 kp

~6 kb


Ladder that has not been denatured sufficiently

Ladder That Has Not Been Denatured Sufficiently


Degraded ladder

Degraded Ladder


Mechanical spikes

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


Enhanced image of mechanical spike

Enhanced Image of Mechanical Spike

Bands are sharp and do not fade like real peaks.

Spike

28S

Notice classical W shape.

18S


Genomic dna contamination

Genomic DNA Contamination

Genomic DNA skewing 28S peak

Additional Genomic DNA Peak

Picture from Agilent Troubleshooting manual


Nanochip contamination

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.


Electrode cartridge contamination

Electrode Cartridge Contamination

Late Migration

Wavy Baseline

Dirty or wet electrode cartridge. Make sure electrode cartridge is clean and dry.


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