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Interpretation of Agilent 2100 Bioanalyzer Data

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Presentation on theme: "Interpretation of Agilent 2100 Bioanalyzer Data"— Presentation transcript:

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.

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