I am trying to detect the fluorescent level of the extracted RNA sample on a 96-well plate. After extraction, I dilute the RNA sample in 20ul solvent. Can I load the 20ul RNA sample directly to the plate or I need to dilute it into 100ul solution to fits the size of the well? And will the different concentration affect the reading?
Hello Yuhao Wan
As far as i know, the final volume that you have in your well DOES affect the reading. That is why you have to tell your software what is the volume/well when you design your protocol. Also, for 96-well plates there is a recommended lower limit for your PCR volume (like 5uL or 10uL).
The amount of RNA eluate that you add in your well depends on your protocol. Commercial PCR kits tell you what is the ratio between RNA sample, mastermix and primers/probes. Generally, you do not need to dilute your extracted RNA in order to fill your well because you generally want your RNA sample to be as concentrated as possible. Also, concentrations will not affect the reading itself, but the kinetics of the reaction and the final outcome (Ct value).
Let's put it short
- generally, you don't have to dilute your RNA sample
- you do not need to fill your well, but you need to have a minimum volume/well
- the volume/well affects the reading, that is why the software asks for your well volume when designing the PCR protocol
- RNA/mix/primers concentration can affect the final result. Diluted RNA samples/mix/primer solution can result in higher Ct values.
Hope this helps!
Definitely, the different concentrations will have different FL intensities.
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