if you are doing your PCR in multiplex format, then your primers are probably competing for a limited amount of reagents. In this case you may try one of the following:

- increase the reaction volume and the reagents will increase proportionally. 

- increase the amount of endogenous control primers while keeping the amount of the target primers at less amount. this will particularly help if you are having a much less Tm for the control primers than the target primers.

if you are doing the PCRs in seperate reactions, then after analyzing the Tms, you probably have to lower the annealing temperature for the control reaction and/or increase the primer concentration.

Good luck

if you are doing your PCR in multiplex format, then your primers are probably competing for a limited amount of reagents. In this case you may try one of the following:

- increase the reaction volume and the reagents will increase proportionally. 

- increase the amount of endogenous control primers while keeping the amount of the target primers at less amount. this will particularly help if you are having a much less Tm for the control primers than the target primers.

if you are doing the PCRs in seperate reactions, then after analyzing the Tms, you probably have to lower the annealing temperature for the control reaction and/or increase the primer concentration.

Good luck

Usually I would first check my primers if I couldn't get bands. Tm, GC content are really important for PCR primers, and extension time is another thing you need to think about. In your case I will suggest you to calculate your Tm again to see if the internal control reactions can be done under similar Tm as your target gene. Also check if the band size for your target gene and internal control PCR are similar. If not, design another primer for similar band size with similar Tm will probably solve the problem.

Try to test other TMs  (make a gradient), check GC and extensión time! Also, you can use dmso to improve pcr reacción .

Did you make some assays with DNA? Or just did it with cDNA? If not, you can make assays with DNA and find the best DNA and MgCl2 concentrations, and Tm. First, it's better that you make assays in separate reactions.

Your PCR has not uptimized because the primers did not bind to the DNA. May I suggest that you check the length and sequence of you primers because they determine to a large extent the melting temperature of the primers. Also check the GC content and salt concentration because high NaOH denatures DNA to ssDNA.

How did you do the reverse transcription? More specifcally, which primers did you use in the RT step? If it was gene specific primers, then you would not get any amplification in your reference genes...

So I have used ProtoScript II First Strand cDNA Synthesis Kit for reverse transcription and used oligo d(T)23 VN in Standard protocol. Moreover I used DNase I Set (Zymoresearch) to digest the DNA. 

Sompop, that should not exclude amplification from the reference genes, unless your reference gene primers are very towards the start of the genes, but actin isn't a very big gene so this should really not be a problem. I assume that you checked the ref primers bind properly to the Arabidopsis sequence. One thing to keep in mind with designing RT-qPCR primers is that if you design them properly (ie one of them spans an exon/exon border), you will not be able to detect certain splice forms that have an alternative exon/exon border. As I have no knowledge of Arabidopsis I could not say if it expresses particular splice forms of Actin, UBQ, Elfa.

Getting good amplification in your RNA of interest suggests that your cDNA is ok. However, I would check if this is specific amplification. I assume you did a water control and -RT control on your sample? If those are clear, I would simply re-do the PCR of your reference genes (simplest explanation is that something went wrong with pipetting - not saying that it's likely, BUT you could spend weeks troubleshooting something that may simply have been a case of pipetting water instead of primers or mixed up forward and reverse primers). From you saying you made a Mastermix - I assume that you made a Mastermix for polymerase+water+template and then added the primer sets?

Or did you do a Mastermix containing all primers? In that case you could do separate reaction for your RNAs.

if you are doing it in a multiplex system , may be the optimised condition for gene of interest is different from what is required for control. I think is better to do separately and then optimise for multiplex system.