Hi, I am not sure if I understand your question correctly, so let me clarify on this first.

You overexpress RNA (in cells) then do PCR. Do you mean you do reverse transcription PCR (then get DNA, of course)? Or do you do in vitro transcription?

In any case, if you are looking for ways how to improve gel extraction kit, there are tricks.

1) QIAgen Gel extraction Kit

2) Make sure the pH of your binding solution is correct (usually indicated by color), when you add isopropanol, make sure you mix all components properly

3) When applying washing solution (the one with ethanol), let the solution incubate with the membrane for 5 minutes before spinning

4) Repeat washing step once or twice to make sure you remove salt from the membrane. The elution efficiency is highly dependent on salt concentration

5) Elute with elution buffer not with water as it is hard to control pure water pH.

6) Prewarm elution solution to 65 - 70 °C, After adding on to the membrane, incubate5 more minutes on the heat block.

7) You can reload the eluted product to the membrane again.

8) Load a lot of DNA/RNA to electrophoresis in the first place. Gel extractions are very inefficient, regardless what manufacturers claim. I usually precipitate my PCR reaction with ethanol, redissolve in small volume and load into small well. It can sometimes allow me to see the DNA without the need of using UV light (also depends on your dye

Or you can alternatively try to use anion exchange FPLC. We use it for DNA, as well as RNA purification.

http://www.ncbi.nlm.nih.gov/pubmed/23929938

I completely agree with Jiri Koubek. I can also say, if you're going to cut a RNA band from agarose, make sure you're using a new, sterile, clean and single use scalpel. It is a important carrying source for RNAses contaminations.

And also I completely agree, the yield is always low extracting from gels, it range from around 20% to 70% depending on size of the fragment, the pH of reagents, the brand of the kit, the additional prewarming step for eluition buffer etc. 

Make sure you're loading on gel a consistent amount of RNA or even cDNA if you're going to perform cDNA synthesis before loading the gel.

Hi there

Hi there

the above is sound advice but I would add a couple more points:

1. Incubating for 5 min with was solution on the column will improve the effectiveness of desalting or the quality of your DNA but has no substantial impact on the recovery or quantity of DNA. One thing that can be done is to use as suggested preheated elution buffer as already suggested but leave that for 5 min on the column before spin elution. In addition recovery can be further improved by performing a second elution as described and then pooling the 2 recovered fractions or taking the first eluate spun from the column reapplying to the column waiting for another 5 min and spinning again

2. In addition melting your PCR product at the maximum recommended temp but got double the time can also improve matters as even tiny amounts of insoluble colloid in what you apply to the column can appreciably compromise purification and Greatly reduce your yield of PCR product

Finally you might want to consider providing you know your PCR reaction is specific not applying to an agarose gel and then extracting from the band but purifying using a column the aqueous PCR reaction itself. This invariably leads to better recovery compared to gel extraction