Why I am having different results in RT-PCR using different cDNA from the same RNA extract?

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Is it high molecular weight or aggregated proteins?

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What is the cause of this problem in western blot?

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

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RNA-seq DEGs downstream analysis and reporting in literature?

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Katie A Burnette

If you made the first and second cDNA separately from each other, it could be differences in RNA stability/degradation over time (freezing & thawing damages RNA), degradation or concentration of reagents in the kit, etc.

You can adjust for these differences using a proper and full set of controls.

Martin Chenal

mRNA is so unstable that I would not recommend doing cDNA synthesis several times on the same sample, unless you properly stored it in single-use aliquots at -80. If you need biological replicates, it is better to start again from the mRNA extraction.

Eman Bassiony

This mostly regarding to the high instability of mRNA. As others advice you, you need to prepare cDNA from a fresh prepared mRNA sample and avoid stored one for cDNA preparation.

Best wishes