Microarray labeling:Direct vs Indirect - (Nov/14/2006 )
Can anybody tell me the difference between direct and indirect labeling and when one should use either technique?
For Gene expression there are 3 labeling methods I tried,
1) "direct labeling" Incorperation of labeled nucleotides
2) "Indirect labeling" Incorperation of Amino modified nucleotide. After the purification you can label the amino modified nucleotides with NHS-dyes
3) "chemical labeling" No incorperation of modified nucleotides but chemically label your target directly
- 1) quick, most common method, the whole sample will be labeled (so real dye swap is difficult), expensive in the case for aRNA. Introduces possibly the largest bias of the 3 methods
- 2) You only have to label the amount you hybridize, possible for dye swap experiments, labeling efficientcy can vary alot (in my hands). Purification of the labeled sample can be a problem (also in my hands)
- 3) Only have to label the amount on the slide, possible dye swap experiments, Need to know exactly the concentration of the sample, need to have very pure cDNA/aRNA, and for cDNA should be clean of RNA otherwise you will also label this.
Have to say that this is my experience, this probably does not count for everybody,
hope this helps