I am trying to quantify and compare genes expression for germination, flowering and fruiting in pearl millet and napier grass using Arabdiopsis thaliana using ABC model.
Answers are :degenerate PCR primers based on Arabidopsis and Oryza sequences. The forward degenerate PCR primers needs to be designed manually looking at the aligned nucleotide sequences of the homologous sequences as submitted in NCBI-GenBank or similar public resources. So if degenerate forward primers based on conserved residues at N-terminal ends of the proteins and oligo(dT)18 reverse primers that are complimentary to the poly-AA Tails in mRMA, then one can get amplicons of varied lengths for different organisms. A little twitching of the PCR conditions using a few gradient PCR experiments would help you amplify much species-specific gene of interest. Also try to use I (Inosine) as a base for positions with 4-fold degeneracy such as A/T/G/C; while grouping the nucleotide sequences (end to end) using Clustal W or any such free multiple sequence alignment tool, would allow you to group the sequences and thus "design several such degenerate PCR primers" to use them taxa-wise (say within plants, or across 2 families in animals, or across several families of plants/ animals). Just order to synthesize more amounts, as with growing fold of degeneracy, the combination of primer mixes yields would be differential ! This approach worked for me zillion times !
Many useful links are there on the web: http://dps.plants.ox.ac.uk/langdalelab/protocols/PCR/degenerate_PCR.pdf and so on. Let me know if you need further details or clarifications. Best wishes.
Degenerated primers are the best option as Biswapriya Biswavas Misra says. If you don't want to make the degenerated primers manually from scratch, I recommend to you CODEHOP software (http://blocks.fhcrc.org/blocks/codehop.html). With this software I was able to obtain degenerated primers for Euclayptus globulus genes based on Arabidopsis, Populus and Vitis sequences without a problem. Give it a try.
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