Hi all,
I have a question regarding proper dilutions and conversions of my RNA.
- According to my protocol, my cDNA kit is optimized for 1ug of RNA and calls for this amount.
- I would like to dilute all of my RNA samples to the same concentration so I can pipette the same amount of RNA into tubes to make cDNA.
- I understand how to dilute the RNA samples (adding x amount of water depending on ng/ul) but what concentration should I dilute them to? This is where I get confused. What is the proper quantity of RNA that I should use for cDNA synthesis? How do I get 1 ug from each sample?
This might be a silly question, but I am getting confused by the conversions and wording. Thank you!
The amount of RNA you can add will be dependent on your cDNA synthesis kit or RT enzyme. For example in many enzymes you will preform the reaction at a final volume of 20µl and will need to add 4µl of buffer, 1µl of primer and 1µl of enzyme which leave you 14µl free for your RNA.
The concentration you prepare your RNA at will depend on the concentration of your RNA prep. I generally bring all my sample to the concentration of the sample with the lowest concentration.
Once you have that you can calculate how much to take in order to have 1µg in each reaction. For example if your concentration is 200ng/µl than you will need to take 5µl in order to have 1µg of RNA.
Yoav Lubelsky Thank you so much! This is incredibly helpful and makes everything much clearer.
Yoav has a good suggestion, you can bring the samples to similar concentrations. If you feel like your samples will give >100ng/uL yield for RNA, using C1V1 = C2V2 formula to dilute your samples to a fixed concentration is useful. Just make sure the volume you intend to take out from the RNA does not exceed the kit's allocation for cDNA conversion volume. That way you always pipette a fixed uL for every sample for the same input, very convenient.
The above answers are both good! Generally, we try to make cDNA using 1ug of RNA in a 20ul reaction, so we have 50ng/ul of cDNA for downstream applications. Usually downstream applications include qPCR, so how much cDNA you'll need depends on how prevalent your primer targets are (i.e if you're looking at something highly expressed like a housekeeping gene, you may want to cut how many ng of template you use so you don't blow out the CTs, but if it is low you want to add more to make sure you catch it and adjust which housekeeping gene you use accordingly).
Remember, do not quantify your cDNA after you make it, this will be inaccurate and overestimate how much you have! You need to base how much template you have on the final concentration post RT. Since one generally does not "clean up" the reaction after going from RNA to cDNA, you still have all those enzymes, buffers, etc that will make your nanodrop quantification much higher than it actually is.
Hope this helps!