Troubleshooting

A. Low Overall Fluorescence Signal Intensity
Consider these suggestions if the average signal intensity from both your sample RNA and the Positive Control miRNA are very low (<500 RFU).

1. Troubleshoot miRNA sample labeling
The most common cause of low overall fluorescence signal is poor labeling of the miRNA sample. Troubleshooting suggestions for the mirVana miRNA Labeling Kit are provided in the Instruction Manual supplied with the product.

2. Excessive exposure of the labeled sample to light
Fluorescent dyes are subject to photobleaching. Exposing them to light, either before or after coupling, will reduce signal intensity. Limit the exposure of fluorescent dyes to light by conducting coupling reactions, labeled miRNA clean-up, array hybridization, and array washing in the dark when possible.

3. The array slide was placed in the scanner incorrectly
Improper placement of slides in the scanner, for example, upside down or backwards, can result in no signal from the scanned region. Mark your slides as you spot them so that orientation can be easily determined later.

4. Hybridization problems

a. Incorrect ratio of miRNA Hybridization Buffer to miRNA sample
The dilution of 3X miRNA Hybridization Buffer with labeled miRNA sample must be precise so that the final concentration is 1X miRNA Hybridization Buffer. This ensures the proper formamide concentration for optimal hybridization stringency.

b. Hybridization and/or wash temperatures were higher than recommended
The recommended hybridization temperature is 42°C. If in doubt, verify the incubator temperature with a calibrated thermometer. Conduct post-hybridization washes at room temperature. If the ambient room temperature in your lab is over 25°C, washing may be too stringent and could result in loss of legitimate signal.

5. Nuclease-contaminated tubes, tips, or equipment
Using pipettte tips, tubes, or other plasticware that is contaminated with nucleases during purification and/or handling of miRNA samples can degrade miRNA, reducing yield and the size of miRNA. Both RNases and DNases can be removed from surfaces using Ambion’s RNaseZap.

1. Partial or complete drying of the hybridization reaction on the array slide

• Exposure to air during washing is a common problem encountered by people attempting array analysis for the first time. When miRNA arrays are exposed to air for more than 1–2 sec, it can allow partial drying of the array and problems with blotches, streaks, and dots of signal that are not associated with probe.

• Make sure that the repositories in the hybridization chamber are filled with 1X miRNA Hybridization Buffer to maintain humidity.

• Make sure that the area under the coverslip is completely filled with miRNA hybridization mixture before starting the hybridization.

• Be sure to check the position of the coverslip over each miRNA array just before sealing the hybridization chamber. If a coverslip gets too close to the edge of the slide, the miRNA hybridization mixture can wick under the slide, leaving the miRNA array with insufficient hybridization mixture.

2. Incomplete washing
If there was a problem with washing the array, then repeating the entire wash procedure may help remove the excess signal from the array. Following are some precautions to observe in subsequent experiments to avoid having to re-wash your array:

• Follow the washing procedure exactly, and be sure to completely submerge the miRNA array in wash buffer at each step.

• Be sure to put the array slides into a clean slide rack when switching them from the Low Stringency Wash to the High Stringency Wash. Otherwise detergent may be carried over, causing detergent residue on the slides.

1. Incomplete reactive dye quenching and removal
If the labeling moiety is not removed by column purification, it can interact nonspecifically with elements on the array. Check the color of the solution of the labeled and purified miRNA. The solution should be clear or lightly tinted. More intense color likely means that uncoupled dye is contaminating the labeled miRNA sample, and it cannot be used for miRNA array hybridization. This could occur if too much fluorescent dye is used in the labeling reaction, or if the glass fiber filter inside the miRNA Labeling Column is not properly positioned and sample bypasses the filter during the purification.

2. Low stringency hybridization conditions
The recommended hybridization temperature is 42°C. Lower temperatures can lead to nonspecific interactions between miRNAs in the sample and probes on the array. If in doubt, verify the incubator temperature with a calibrated thermometer.

3. Total RNA was used to prepare labeled miRNA sample
Labeling total RNA can lead to nonspecific binding to the relatively short probes used for miRNAs. We recommend purifying miRNAs using the flashPAGE Fractionator before labeling to eliminate non-miRNAs from the sample before analysis with the mirVana miRNA Array System. Alternatively miRNA can be isolated using traditional gel electrophoresis [protocol].