A lot of RT buffers contain things like dithiothreitol (DTT) which can precipitate when buffer is frozen: it's usually a good idea to mix (pipette up and down in fine) your cDNA synthesis buffer well before use.

I think this is unlikely to be RNA secondary structure + precipitation: these would be truly gigantic aggregates if so. Precipitating RNA just tends to look like a very slightly cloudy solution: you almost never have enough RNA present, and those RNA molecules are almost never long enough, to get large, visible aggregates.

Hi Raunak Jahan, Before commenting on precipitation in the cDNA product, may I know the RNA concentration you obtained after RNA isolation and also would like to know how you checked the RNA quality. I generally doubt my RNA if it shows a very high concentration, also I always run the agarose gel on my RNA sample before proceeding for cDNA synthesis. I may doubt the contamination in the RNA sample here.

Thank you Dr. John Hildyard .If you think the Rt buffer would be the issue, I have the following concern. I purify the RNA through PCI extraction. After extracting the RNA from ethanol, if some leftover unseen ethanol is present in my RNA pellets, could those ethanol be responsible for this

type of aggregation? Please, share your thoughts.

Thanks

No, ethanol would not do anything of note, and the probability of 'leftover ethanol' being present should be zero.

Also, you state that your concentration is "very high, 3750ug" but this is an amount, not a concentration. 3750ug per what?

It would probably help, to be honest, if you provided a lot more detail about what you're doing at each step, and why you're doing it that way.