First of all it is important for others to know your setting.

Genomic DNA, cDNA, bisulfite conv. DNA or RNA ?

Let's say I need to do real time PCR to quantify the cDNA(which is going to happen sooner), what should I be care for??

You must follow 6 criteria

1. Primers should be between 18 to 30 base long.

2. Primers should have a Tm value between 55-72 degree C.

3. The Tm value of each primers designed for one micro-satellite should be within 5 Degree C of one another.

4. Primers should have G/C concentration between 40-60%.

5. Primers should never repeat a base four or more times in a row.

6. Primers should not have more then 2 G or C bases within the last 5 bases on their 3' end.

And is there anything I need to be precaution about the reading frame??

hi,..well said by banwari. inaddition do check the homology of ur primer using BLAST, to prevent non-specific bindings...

Depending on your reverse transcription protocol you should take care where you place your amplicon. If you use oligodT-18 primed reverse transcription it is advisable to place your amplicon no further than 2000 bp upstream from the end of the polyA-tail of your transcript if you use a standard reverse transcriptase like M-MLV since the RT reaction does not go much further than that and you miss copies otherwise. If you use something like Superscript or other "full-length"-RT this does not matter.

A very good tool for primer design is the primer-blast available from the ncbi website. you can simply input the accession number of the gene and specify the search parameters like length, gc and everything else the people suggested above (see "advanced parameters"). The tool uses primer3 in the background and gives very good results from my experience, but you should input the right salt and oligo concentrations for the full potential of the tool . One more advice - design, if possible, all primers to have one specific temperature, e.g. 60°C. By doing so you can run sybr green reactions for multiple (well expressed) genes on the same plate, thereby saving material and time.