Hi, 

Your question is a little confusing. Are you saying that the qPCR of your selected genes did not show any amplification (eg. no Cq value)? If so, you may need to provide more details such as the quality and integrity of your RNA samples and information about your PCR reaction so that others can help to address your issue.

Thanks. 

Actually i am unable to detect where is the problem? But i doubt its in the RNA preparation of my control. i Feel like cells are not lysed properly. So can you tell me if macrophage get lysed in trizol upon pipetting or it need to be sonicated.

Hi

it seem you have problem in RNA extraction. first I would leave your pellet with trizol for over night in -20 this might give you better yield of RNA. second I would be careful with last step and try to eliminate all ethanol from your samples. also I would recommended to use Qiagen RNeasy Mini Kit for good quality of RNA. the best way to measure the RNA quality is by using bioanalyzer which is give you RNA integrity number. however, it is expensive a bit.

I hope this will help

good luck

Thanks  @ Massar Alsamraae

Trizol should be able to lyse macrophages. Just pipette it repeatedly or vortex the mixture until you do not see any big chunks of tissues. If you think the problem lies in the extraction, OD260/280 and OD260/230 as well as a simple agarose gel electrophoresis can provide valuable information about possible contaminants, purity and integrity of your RNA samples. You may provide those information here so that we can see if extraction is the problem.

Additionally, I find the amount of sample used for extraction will greatly affect the quality and quantity of your RNA output. For example, if you use too much sample in a column-based extraction kit, you may risk overloading the column and get contaminated RNA. Generally, for trizol, you may want to keep a ratio of 1mL trizol per 50-100mg of tissues. Do scale up the volume of trizol accordingly if you put in more samples.

Lastly, as Massar has mentioned, a bioanalyzer can be used to determine the RNA integrity. As for concentration measurement, a qubit fluorometer will give a more accurate result compared to Nanodrop. Both bioanalyser and qubit are, however, much more expensive per assay compared to agarose gel electrophoresis and Nanodrop respectively. Personally, I don't think they are necessary for qPCR assay. 

I hope this helps.

Hi,

I personally use the Agilent 2100, it's similar to gel electrophoresis but microfluidic-based. I will also tell you the total RNA concentration in your sample.

Hope it helps