Hello!
I usually use 2μg of extracted RNA to prepare cDNA using "Thermo Fisher high capacity cDNA reverse transcription kit" and proceed with qPCR.
I was wondering if anyone has ever used a higher amount (for e.g 3μg or 4μg of extracted RNA) and saw significant changes in the Ct values?
Thanks in advance for your help!
You shouldn't use more than the kit can handle. They have an upper limit because at some point, the reaction is saturated.
And I've never used cDNA "neat". You always need to dilute (unless your gene of interest has really, really low expression).
Good luck!
There is a maximum limit to any kit like Takara 1st strand prime script cDNA kit which has a max 5ug capacity. Also, we must dilute the cDNA because qPCR is a sensitive reaction and so a high cDNA concentration will saturate the reaction. In terms of primer efficiency, RNAase inhibitor, and variation of copy number of a transcript matter a lot for qPCR gene expression.
Why to dilute cDNA, then read this discussion thread.
https://www.researchgate.net/post/Is-cDNA-from-1ug-RNA-diluted-18-equivalent-to-cDNA-from-025ug-RNA-diluted-12
https://www.researchgate.net/post/How_to_proceed_for_dilution_of_cDNA_How_we_can_find_out_quantity_and_quality_of_cDNA
https://www.researchgate.net/post/is_it_necessary_to_dilute_cDNA_before_qPCR_How_much_dilution_should_be_made_How_to_determine_best_dilution_factor
https://www.researchgate.net/post/Why_is_necessary_to_dilute_cDNA_before_performing_PCR
https://www.researchgate.net/post/How_to_optimize_RTqPCR_if_cDNA_dilutions_resulted_in_similar_Cq
I use 1ug with the same high capacity cDNA synthesis kit. More than 2ug is not recommended as you will not be able to ensure 100% conversion of RNA to cDNA. Is there a specific reason for wanting to use higher RNA amount?
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