Amount of RNA for cDNA

[newbie17425]

Hi,

 

I know this question must have been answered before but i would like to confirm it again.

 

I am using Thermo-scientific cDNA synthesis kit and it says take 1pg - 5ug of your template for cDNA synthesis. I am having a RNA yield of 19 ng/ul (micro-dissected) brain sample i.e. the hippocampus with a total volume of 50 ul. I then treat it for DNAse. So, my total volume is not more than 55 ng/ul and i want to save some RNA for future experiments.

 

Now, to calculate 1 ug,

x ul = (1 ug)/(25ng/ul)

x ul = 1000/19

x = 52.63 ul

 

Until now i was using about 2 ul of the template to synthesis cDNA. Will it be fine with 2 ul or should i inc. the volume to 50 ul for cDNA synthesis and then proceed on with the qPCR? Although the cDNA synthesis kit gives a final volume of 20 ul after addition of the template.

 

Thanks

[CharonY]

 

 

So, my total volume is not more than 55 ng/ul and i want to save some RNA for future experiments.

 

I am not sure where you got, this is a concentration not a volume. According to your values you have maximum 50x19=950 ng total RNA. Unless you mean that you added 5ul reaction buffer for the DNAse treatment, which leaves you with the same amount of RNA but now it is in 55 ul.

 

In either case, your total will still be 0.95 ug (unless I misunderstood something), i.e. lower than you calculate with. So something is off.

 

In either case, for critical applications I recommend an accurate determination (i.e. no nanodrop) after DNAse treatment as this is going to be your final yield and it can be substantially different, if the prep was not optimal.

[newbie17425]

 

I am not sure where you got, this is a concentration not a volume. According to your values you have maximum 50x19=950 ng total RNA. Unless you mean that you added 5ul reaction buffer for the DNAse treatment, which leaves you with the same amount of RNA but now it is in 55 ul.

 

Yes, i meant after adding the DNAse treatment but yes i forgot that won't change the concentration but only the volume.

 

How did you calculate that i have 0.95 ug?

 

What do you mean by accurate determination after DNAse treatment?

[CharonY]

50 ul x 19 ng/ul = 950 ng.

 

I mean that after DNAse treatment you make a RNA concentration assay (it seems that you did before), Spectroscopic (i.e. UV) is fine if your sample is clean but requires a bit of volume to be accurate. Better assays (in most hands) are fluorescence based, including e.g. Qubit or Bioanalyzer systems.

[newbie17425]

I never did. I extracted the RNA and checked for its purity on nanodrop. Should i again check after DNAse treatment?

 

Also, how much of this DNAse treated RNA should i take for the cDNA synthesis. I take 2ul (conisdering the yield to be 19 ng/ul) for the cDNA synthesis and recommended range is 1pg - 5ug.

[CharonY]

Well, the first thing you should do is figuring out how much RNA you actually have post-digest. Anything else would be guessing.