2 Intact Total RNADistinct 28S ribosomal subunit (or prok. 23S): ideally 2X size of 18SDistinct 18S ribosomal subunit (or prok. 16S)28S18SNo well defined peaks between ribosomal peaksMarkerFlat baseline throughout electropherogram~100 bpMarkerSmall 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 intensitiesThe 28S subunit often degrades firstIncrease in intensity ofsmaller digested RNA fragmentsBaseline between and to the leftof ribosomal peaks becomes jagged
4 Partially Digested Total RNA Using Trizol Extraction Decrease in ribosomal peak intensities~100 bpmarkerDigested RNACombination of 5S, 5.8S, tRNAs, and an increase in digested RNAs
5 Heavily Digested RNASamples of this quality, if labeled and hybed to a chip, might be in question.~100 bpPeaks shift to leftHigh digested RNA peaks
6 Heavily Digested RNA Using a Hot Phenol with Beads Extraction
8 Characteristics of Good Labeled cRNA ~1 kbMarkerRound Curve with a few small peaksSmearSmall initial hump
9 Labeling with Partial Fragmentation Labeled cRNA with this image are OK to fragment and hyb, but not without risk.~1 kbMore 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 bpMarker
12 Low Quantities of Fragmented cRNA This sample may still be OK to hyb, but frequent failures have been reported withcRNA 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 bpNotice that the gel image looks normal.
16 Mechanical SpikesThese 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.Mechanical spike
17 Enhanced Image of Mechanical Spike Bands are sharp and do not fade like real peaks.Spike28SNotice classical W shape.18S
18 Genomic DNA Contamination Genomic DNA skewing 28S peakAdditional Genomic DNA PeakPicture 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 MigrationWavy BaselineDirty or wet electrode cartridge. Make sure electrode cartridge is clean and dry.