cDNA-AFLP®
Gene Expression Profiling
The cDNA-AFLP technology permits the display and quantification of transcripts based on AFLP fingerprinting of double-stranded cDNA. The transcript profiles obtained using this technique, are a reliable and efficient tool for the identification of differentially expressed mRNAs.
How it works
The fingerprinting procedure consists of the following steps:
- isolation of mRNA from the samples of interest;
- reverse transcription of mRNA using an oligo-dT primer to produce cDNA;
- digestion of double stranded cDNA with a pair of restriction enzymes;
- ligation of adapters specific for the two restriction sites;
- pre-amplification of fragments with primers specific to the two adapter sequences;
- selective amplification with adapter specific primer combinations with nucleotide extensions at their 3’ ends (usually varying between 1 and 3 selective nucleotides);
- visualization of individual fragments on a polyacrylamide gel (using radioactive gels: one of the two primers is end labeled with 33P);
- Analysis by image-processing software. This software identifies the cDNA-AFLP fragments and quantifies the intensities that correspond to the original expression level.
Figure 1. An example and a detail of a cDNA-AFLP fingerprint that shows up and down regulation as well as constant expression in two samples.
Benefits
- it can visualize over 90% of all expressed genes;
- no require prior knowledge of the genetic sequence necessary of the analyzed organism / genes;
- since it is PCR based, it is very sensitive and can detect down to 1 mRNA molecule per cell;
- bands corresponding to genes / expression profiles of interest can be excised and sequenced;
- in case the sequence of relevant genes is known, the fragment size and gel position of the resulting cDNA-AFLP fragments can be predicted. As such the gene expression of known genes can be specifically followed.
Applications
- Gene Expression Profiling;
- Biomarker Identification;
- Promoter Identification;
- Targeted EST Sequencing.