Dear Debaashish, 

I will recommend to run housekeeping gen/reference gen along with your sample. At least you can exclude master mix, polymerase etc if your sample has no detectable Ct-value. Furthermore RNA quality and primer design is also important to keep in mind. Can you tell more about the origin of your unknown sample?

Good luck and all the best!

Greets,

Tse

We always keep a positive control and negative control for PCR. You can keep a positive control with known copies of DNA. Be careful about handling of positive control DNA. Contamination of your reagents with positive control may lead to amplification in test as well as negative controls leading to useless data. Further, it is difficult to find the source of contamination.

Keep a positive control but handle it with care. 

I would recommend a pilot experiment to optimize the conditions (master mix, cycling conditions, primer efficiency and other parameters) prior to running your unknown samples. Due to the potential for contamination when including a positive control (as in Dr. Rao's answer above) I would avoid including it in your final experiment. Definitely include the housekeeping gene, though, both as a positive control for the reaction and potentially as a normalizer.

It is recommended to keep positive control and negetive/water control. 

It is good to use positive control along with House keeping genes, specially if you treat the cells (either stimulate with reagents or knock down). It will help you to identify any error in the process (if you don't see any Ct value for your unknown samples)