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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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



Completely digested rna
Completely Digested RNA Extraction

~100 bp

Marker


Characteristics of good labeled crna
Characteristics of Good Labeled cRNA Extraction

~1 kb

Marker

Round Curve with a few small peaks

Smear

Small initial hump


Labeling with partial fragmentation
Labeling with Partial Fragmentation Extraction

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 Extraction

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

Peaks still present, but digested


Properly fragmented crna
Properly Fragmented cRNA Extraction

Fragmented samples with this image are ready for hybridization.

~100 bp

Marker


Low quantities of fragmented crna
Low Quantities of Fragmented cRNA Extraction

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 Extraction

200 bp

25 bp

2 kb

500 bp

1 kb

4 kp

~6 kb



Degraded ladder
Degraded Ladder Extraction


Mechanical spikes
Mechanical Spikes Extraction

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 Extraction

Bands are sharp and do not fade like real peaks.

Spike

28S

Notice classical W shape.

18S


Genomic dna contamination
Genomic DNA Contamination Extraction

Genomic DNA skewing 28S peak

Additional Genomic DNA Peak

Picture from Agilent Troubleshooting manual


Nanochip contamination
Nanochip Contamination Extraction

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 Extraction

Late Migration

Wavy Baseline

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


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