Hi Zhang

Just to be clear: Are you saying that your primers yielded a band from the genomic DNA but not from cDNA. If so there are a plethora of reasons why this might be so but you need to be a bit more specific provide additional information/data with your question

Then I might well be able to help you :-)

Regards

Have you used the same cDNA to amplify a housekeeping gene?

Dr Moritz ,Dr  Laurence and Dr Eman.

Thank you for all of your help. The answers is very useful for me.but I am sorry that  i  don't know how to reply to you under your answers....i can't find the dialog box to reply.

• I used an ACTIN gene to amplify, but  the pcr product of the Cdna and the DNA are the same size.
• yes, my primers yielded a band from the genomic DNA but not from cDNA. While i designed the primers from cDNA. 
• Best  Zhang

Getting a band from your actin from the cDNA preparation means that you have a problem with your gene under investigation. Check first that your gene is transcribed and if yes try to design primers that flank intron-exon regions to be able to eliminate possible contamination with genomic DNA.

I agree with Eman

If you have managed to amplify a product from your genomic DNA but not your cDNA that suggests that their is something not quite right with your cDNA and/or RNA, i.e. degradation of your RNA and/or salt contamination, indicated by a 260/230 ratio of < 1.0. See a previous answer of mine:

Any suggestions about a specific primer in RT-PCR which gave very clear and specific band several times but stopped working? - ResearchGate. Available from: https://www.researchgate.net/post/Any_suggestions_about_a_specific_primer_in_RT-PCR_which_gave_very_clear_and_specific_band_several_times_but_stopped_working [accessed Nov 18, 2015].

Think about the following:

1. What is the quality of your RNA and in particular what is the 260/230 ratio: < 1.0 indicates the presence of salts (or trizol for organic extractions) and in particular GITC. This salt is present in Trizol and RNA column purification lysis buffers and also the first kit wash buffer (- ethanol). In lysis buffer it is designed to inhibit RNAses and provide a stable environment. In the first wash buffer it is present to denature any protein remaining on column prior to RNA elution. It can however bleed through to final eluted RNA. This is indicated by 260/230 ratio less than 1.0. If so it inhibits down stream application including RT. Thus is your 260/230 ratio is < 1.0 this might explain why you obtained no amplification from your cDNA. To remove GITC from an RNA prep either resuspend in an equal volume of 70% ethanol and repeat column purification or  precipitate RNA with 3 volumes of ethanol; 1/10 3M sodium acetate pH 4-5 @ -80C overnight or for 1 hour. Spin 13k (microfuge) for 20 min then wash with 70% ethanol to desalt and remove GITC. Better still add 1ul of 1mg/ml to 20mg/ml glycogen to pellet before precipitating. This will render the pellet visible and enable you to wash (desalt) by vortexing without losing your RNA pellet

2. Is your RNA degraded ? Check by running 100-500ng on an Agilent biochip or the same amount on a 0.7% agarose gel. add 10% formamide to sample and denature @ 90C for 2 min then place on ice before running (with loading dye)

3. Did you design your primers to an internal exon ? If so you will selectively amplify from better quality and/or quantity of genomic DNA. Instead as mentioned target exon-exon boundaries using primers designed with primer BLAST to NCBI RNA refSeq (=NMXXXX). I have attached a detailed protocol for you to follow which tells you exactly how to do this. Primer composition is just as important as primer location and I have enunciated a few simple rules in the second word doc supplied :

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

4. How much RNA did you perform RT with: Try using 500ng to 1ug for 1 hour @ 55C (to navigate secondary structure) and use a thermostable RT polymersase like superscript III/IV or sensi fast (for example). Do not perform RT with < 200ng of total RNA, especially if quality is low ( 260/230 < 1.0) and gel/biochip indicates degradation

5. Especially for degraded RNA bias your primers to the 3' end of the mRNA sequence; that is keep within 1-2kb of Poly A site. Even with un degraded high quality RNA this is essential if you prime with Oligo DT: cDNA is made from the 3'  end of the message so representation is better @ the 3' end, especially with degradation so primers cited > 2kb from the Poly A tail will culminate in poor RT and thus low PCR efficiency. Better still use random hexamers instead of or in addition to Oligo dT during RT

 Your advice is very important for me! Thank you.

Best wishes 

You can try with different dilutions of cDNA