Dear Brian!

Please You skip in the link:

https://www.researchgate.net/post/Im-doing-cDNA-synthesis-with-my-total-RNA-and-its-concentration-is-too-low-Can-I-do-it-if-I-concentrate-using-centrifugation

To concentrate the cDNA, you will need to precipitate it first. You can try to (1) precipitate the cDNA with ethanol/isopropanol or (2) dry the whole eluent in a chilled vacuum evaporator (eg. SpeedVac) and dissolve the pellet/dried sample in the minimum volume of water/TE to have a higher concentration

-Check this protocol for precipitation with ethanol (if you have to precipitate it use glycogen, instead of NA acetate)

https://agctsequencing.wordpress.com/2012/07/06/protocol-alternative-method-for-sodium-acetate-to-ethanol-precipitate-dna/

The only limitation depends on weather you eluted your sample with water or elution buffer. If you have used elution buffer (usually TE) for elution, it will cause salt precipitation thus you will need extensive washing with room T°C 70% ethanol to remove the salt and break any salt-induced DNA aggregates.

However, I recommend checking your synthesizes cDNA first. Perhaps you will not need to concentrate it. The most important is to avoid DNA precipitation as much as you can. It is risky for the homogeneity of the dissolved DNA. Sometimes, a DNA pellet, especially salt-precipitated DNA, when quite dry, cannot dissolve well. As a rule, using precipitated DNA in fine biochemical experiments is almost as horrible as using TCA precipitated proteins. Of course, precipitated DNA is OK for routine, molecular biology – cloning into vectors etc., experiments.

I am a bit confused. Why would you do two separate cDNA reactions? If you are desperate to make twice as much cDNA, why not just do one, double-sized, reaction instead?

I would also not attempt to concentrate my cDNA preps: I always dilute my preps because cDNA synthesis buffer components can inhibit qPCR.

Lyophilising your cDNA will give you a super concentrated mix of inhibitory components (you don't want that), while precipitating your cDNA will likely lose you more than you gain: you will need to run your qPCR in triplicate, and include reference genes, so you're already looking at 20ul+ of cDNA, which is what most cDNA synthesis preps produce anyway. If you precipitate that you might remove some of the inhibitory components, giving you a 'cleaner' 20ul stock, but you'll lose some cDNA in the process.

Honestly, it's hard to answer this more comprehensively without more information. For example, when you say "low amounts of RNA", how low are you talking? And what are your 260/230 ratios? And what is your GOI, and how abundant do you expect it to be?

The most important thing to be sure of is that your cDNA synthesis actually works: with low [RNA] you'll be adding larger volumes of your RNA prep, and thus guanidium salt contamination (which 260/230 checks for) becomes critical. Too much chaotropic salt and all your reverse transcriptases denature.

If your 260/230 ratios are good, then just use as much RNA as you can and see what expression level you observe: make informed decisions after this.

You really don't need much RNA to get measurable, quantitative data via qPCR: it's a very, very sensitive technique.