dear Jaya,

did you mean your dna target and negative control look similar pattern?

Yep, the quantity of DNA template might be causing the problem. You have to be more careful when measure the DNA template concentration. by looking at yours, 1 ug is too much for me as template for qPCR. I usually use 20-50 ng/uL DNA concentration for PCR, and this amount work well. If too much DNA applied, the primers is difficult to be attached in template and there will be no amplification.

So, I recommend you to measure more carefully your DNA template once again and pay attention on dilution factor. it's little bit mathematically. I recommend you read a book 'Calculation for Molecular Biology and Biotechnology' to get more easy understanding.

I Hope it'll help you,

Thanks

Your qPCR concentrations look fine and normally shouldn't affect the results (if the primers are correct for the qPCR to reflect expression levels of the target transcripts). Maybe your gene responds much more to shRNA on a protein level or maybe it is some off-target effect. Did you look at 18s RNA as a reference?

Although, when starting from 1 ug RNA/20 ul, we usually dilute 1:10 (or 1:5) and then use 2 ul of this dilution per 20 ul qPCR reaction. This is still more than enough for typical genes, makes material for more reactions and sometimes reduces artifacts.

Also, it may be a little better to use 0.1-0.5 ug RNA (per 20 ul) in a standard RT reaction for a better linearity of conversion RNA-->cDNA. But 1ug is usually ok for most practical purposes.

http://www3.appliedbiosystems.com/cms/groups/portal/documents/generaldocuments/cms_075428.pdf

http://www.thermofisher.com/ru/ru/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/abnormal-amplification-curves/amplification-occurs-later/your-reverse-transcription-may-not-be-optimal.html

from your question i feel you are doing a relative quantitation experiment.

Now many at times your endogenous control that is like reference genes like actin might give more fold level expression than  your target gene, or sometimes like in your case u r getting same fold expression, dont worry about it, but practically you can do a serial dilution of sample and find in which concentration dont hide the expression of target gene

Dear Jaya:

Fom the informations that you exposed I tell you two things. First, incongruences between transcript accumulation and protein sinthesis have been described for many genes, in your case is very important that you look the melting curves fron your genes, and check the posibility to detect more than one product for your experimental gene. This phenomena is common in gene families and the posibility of detect more than one member depends of your primes design, the amplicon must be correspond to 5' or 3'-UTR, sometimes if one of your primer fall into coding sequence is enough for cross amplification of more than one family member

regards

Carlos

Thank you all for your input