Sample Labelling and Hybridisation
There are many different labelling techniques available, however, the labelling technique to be described here is the direct labelling technique for fluorescent cDNA targets.
Major tips for sample labelling
- Do not label RNA that is not pure, not fully intact and with an unknown quantity
- Avoid enzymatic degradation of the RNA sample such as RNases during labelling and keep the sample on ice
- Protect both fluorescent CyDye reagents from light during both handling and storage
Hybridisation of the samples on the array is the next step. There are two methods for performing hybridisation, manually or using an automated machine. In our laboratory, hybridisation is performed manually.
Major tips for hybridisations
- Do not wear powder treated gloves while performing hybridisation
- Avoid placing the DNA slide on dirty surfaces, as dirt on the rear of slide will affect the result on the front
- Avoid touching the slide surface (spotted area) when transferring the sample mixture onto the slide
- Use exact amount of each labelled sample to obtain reliable data
- The formation of air bubbles is possible and should be avoided. Coverslips should be lifted to get rid of air bubbles
- Choose excellent stringent washes to prevent false and positive signals
RNA Hybridisation Protocol (pdf 10kb)
DNA Hybridisation Protocol (pdf 8kb)