RNA

Common Phenotypes and Solutions - RNA

Additional Samples or Ladder Peaks -- Small peaks next to larger ones, etc

Most Probable Causes

• Gel-dye mix expired -- Use prepared gel-dye mix within one day
• RNA sample not denatured properly -- Heat sample at 70C for 2 minutes
• Particles in tubes -- For reagent preparation, use tubes supplied with the kit (not autoclaved)
• Chip contaminated -- Wear powder-free gloves only

• Dust particles in separation channels -- Don't touch the wells of the chip; clean the electrodes; load the chip immediately after

  taking it out of its sealed bag

Probable Causes

• Dye agglomerates present in the gel-dye mix -- Use dye concentration according to the Reagent Kit Guide
• Chip preparation with cold reagents/chips -- Prepare a new chip

Least Probable Causes

• RNA ladder/sample degraded -- Always wear gloves when handling chips/RNA samples to prevent them from getting contaminated

Degraded RNA Ladder and/or Samples -- No or too few peaks

Most Probable Causes

• RNase contamination of the pin-set -- Decontaminate the pin-set
• RNase contamination of chips and/or reagents -- Use a new chip and/or fresh reagents; wear powder-free gloves when preparing chip

Spikes -- Sharp up and down peaks together

Most Probable Causes

• Vibration of the bioanalyzer -- Don't touch the bioanalyzer during a run; remove vibration devices from bench
• Particles in tubes -- For reagent preparation, use tubes supplied with the kit (not autoclaved)

• Chip/gel-dye mix contaminated -- Prepare new chip with new gel-dye mix; wear powder-free gloves only; don't touch the underside of

  the chip; don't touch the wells of the chip; clean the electrodes; load the chip immediately after taking it out of its sealed bag

Probable Causes

• Dye agglomerates present in the gel-dye mix -- Use dye concentration according to the Reagent Guide

Least Probable Causes

• Power outlett -- Install power filter

Low Sensitivity -- Entire electropherogram is small, squiggly up and down "peaks"

Most Probable Causes

• Gel-dye mix expired -- Use prepared gel-dye mix within one day
• Dye concentration too low -- Use dye concentration according to the Reagent Kit Guide
• Pipetting error during preparation of mixtures -- Check dilution procedure and calibration of pipette
• Chip pipetting error -- Pipette new chip; always insert the pipette tip to the bottom of the well when dispensing the liquid

Probable Causes

• Fingerprint on focusing lens or on the backside of the chip -- Clean lens
• Insufficient vortexing of chip -- Vortex chip for 1 minute; only use IKA shaker for chip vortexing; adjust speed to 2400 rpm

Least Probable Causes

• Autofocus or laser failure -- Check autofocus and laser using the "Hardware Diagnostics"

Baseline Noise -- Baseline very squiggly with many more acute up and down peaks

Most Probable Causes

• Fingerprint on focusing lens or on the backside of the chip -- Clean lens; do not touch the underside of the chip

• Chip contaminated -- Wear powder-free gloves only; don't touch the underside of the chip; don't touch the wells of the chip; clean

  the electrodes; load the chip immediately after taking it out of its sealed bag

• Vibration of the bioanalyzer -- Don't touch the bioanalyzer during a run; remove vibration devices from the bench

Probable Causes

• Dye concentration too low -- Use dye concentration according to the RNA Reagent Kit Guide

Broad Peaks -- Large "hill" or "hump"

Most Probable Causes

• Sample contaminated with genomic DNA -- Check RNA-isolation protocol; remove gDNA and perform DNase treatment
• Leaks current due to contaminated pin-set -- Clean the pin-set of the electrode cartridge
• Electrodes contaminated with RNases -- Clean electrodes with RNaseZap

Probable Causes

• Clogged gasket and plastic adapter of priming station -- Check the priming station as described in the seal test of the manual;

  clean/replace gasket and plastic adapter if necessary

• Wrong settings of the chip priming station -- Check if chip and base plate of priming station are in the right position

Least Probable Causes

• Dye concentration too high -- Use dye concentration according to the Reagent Kit Guide

Missing Peaks -- Noisy and non-straight baseline

Most Probable Causes

• Too high salt concentration in sample -- Refer to the maximum sample buffer salt concentration as specified in the reagent kit

  guide; dilute samples with deionized RNase-free water if necessary

• Clogged gasket and plastic adapter of priming station -- Check chip priming station as described in the seal test of the manual;

  clean/replace gasket and plastic adapter if necessary

• Wrong settings of the chip priming station -- Check if chip and base plate of priming station are in the right position

• Leak current due to contaminated electrodes or wet chip surface (detergents in RNA elution buffer) -- Clean electrodes with

  analysis-grade water and a toothbrush; prepare a new chip, lower vortex speed or mix samples manually

Probable Causes

• Laser broken -- Perform Laser/LED/Optics test and Autofocus test

Least Probable Causes

• Autofocus failure or high voltage power supply defective -- Check autofocus and high voltage power supply by means of the

  "Hardware Diagnostics"

• Sample wells filled with water -- Refer to the Reagent Kit Guide for proper preparation of the chip

Missing RNA Fragment -- Only one distinct peak and marker peak observed

Most Probable Causes

• Too high salt concentration in sample -- Refer to the maximum sample buffer salt concentration as specified in the reagen kit

  guide; dilute samples with deionized RNase-free water if necessary

Probable Causes

• Sample degradation because of RNase-contamination of electrodes or reagents -- Clean electrodes with RNaseZap; use a new chip

  and/or fresh reagents; wear powder-free gloves when preparing the chip

• Incompatible column material of RNA extraction kit -- Use RNA extraction kit of different vendor

Wavy Baseline -- Severe waviness of the baseline (much like a sinoid curve)

Most Probable Causes

• Following sample contaminated with gDNA causes baseline fluctuations -- Check RNA-isolation protocol; to remove gDNA, perform

  DNase treatment

• Leak current due to contaminated electrodes or wet chip surface (detergents in RNA elution buffer) -- Clean electrodes with

  analysis-grade water and a toothbrush; prepare a new chip and lower the vortexing speed or mix samples manually

• Clogged gasket and plastic adapter of priming station -- Check chip priming station for seal test; clean/replace gasket and

  plastic adapter if necessary

• Contaminated electrode cartridge -- Clean the electrode cartridge

Probable Causes

• Wrong settings of the chip priming station -- Check if clip and base plate of priming station are in the right position

Least Probable Causes

• Autofocus failure of high voltage power supply defective -- Check autofocus and high voltage power supply

Cross contamination -- Unexpected peaks

Most Probable Causes

• Contamination of pipette tips -- Use fresh tips for each pipetting step
• Contamination of electrodes -- Clean electrodes between runs

Least Probable Causes

• Leaks currents due to contaminated pin-set -- Clean the pin-set of the electrode cartridge

Late Migration of the RNA Ladder or Sample

Most Probable Causes

• Vortex speed too high -- Vortex at lower (medium) speed; for chips use only the IKA vortexer
• Leak current due to dirty electrodes -- Clean electrodes

• Detergents in RNA elution buffer lowers surface tension in wells of chips leading to liquid spill on top of the chip during

  vortexing -- Prepare a new chip and lower the vortex speed or mix samples manually

Probable Causes

• Vortex adapter not connected tightly -- Press vortex adapter tightly on mount; replace vortex adapter

• Clogged gasket and plastic adapter of priming station -- Check chip priming station with seal test; clean/replace gasket and

  plastic adapter if necessary

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