How can a higher yield of RNA contribute to a better cDNA synthesis when conducting a RT-qPCR?

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Why do the PCR bands from the cDNA templates (extraction from the cell after transfection) show different size from the band of the plasmid template?

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What is the best concentration of cDNA to be used for qPCR?

I have synthesised cDNA from 500ng/ul of RNA. The concentration of the cDNA Is about 5000ng/ul. Should I dilute my cDNA or just use it as it is for my qPCR? I usually use 2ul of cDNA for a 20ul...

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Krishna H Goyani

You can refer this PDF might be it helpful.

https://genome.cshlp.org/content/2/1/86.full.pdf

Péter Gyarmati

Well, your RT will go on as long as you have primers/random hexamers available. With that said, you will need around 25-50ng of cDNA for your PCR, more DNA will not do much good.

Chayan Bhattacharjee

Normally all the commercially available cDNA synthesis kits are very efficient. You can get a good quantity of cDNA from even 50 ng of RNA. But for cDNA synthesis, you should be more concerned about the quality of your RNA than quantity. The amount of RNA will only matter if you are dealing with transcripts that are low in copy number or have low expression.