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Can anyone point me research papers using Superscript iii on <200ng RNA?


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#1 Tristen

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Posted 30 May 2018 - 08:27 PM

The invitrogen protocol says 1pg is sufficient. All the papers I find either don't say how much RNA they started with, or they used 1ug or more. I have around 150ng bacterial RNA, and so am using random primers to synthesize the cDNA. The 260/280 readings are close to 2. I need to make a case from the literature before I can move forward with the cDNA synthesis.

 

Thanks in advance for any help.



#2 OldCloner

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Posted 08 June 2018 - 11:36 AM

Tristen-

 

It matters a great deal what you want to do with your cDNA afterwards. If you are, for example, doing qPCR on one target, you will need less cDNA than if you are qPCR-ing 10 targets. Or more.  Figure out how many doses of cDNA you will need for your downstream experiment (consult the directions for that experiment, your Sybr green kit or whatever you are using, for how much cDNA should be put in each reaction). Then multiply by how many reactions you need to do (how many gene targets plus controls) Add "extra" for safety, then you will know how much cDNA you will need from that sample.  You can usually assume with random primers that you will generate an equal amount of cDNA as the amount of RNA you started with; its a fuzzy assumption-but just go with it. 

 

Then read the Superscript instructions- they say you can start with a range of RNA from 1pg to 5 ug in one 20 ul rxn.  Does the total amount of cDNA you will need fall within that range?  If so, a 20 ul rxn will be big enough.  If you need more, plan to scale up from a maximum rxn (I.e. using the max amount, 5 ug, of starting material). Example: you calculated that you need 10 ug of cDNA to do all the qPCR targets of interest (plus extra), so do a 40 ul first-strand synthesis with 10 ug RNA starting material.

 

You can just go ahead and convert all your 150 ng RNA to cDNA in one Superscript rxn- and then worry about how much of that to use in your next step. You may not need all of the cDNA you made, depending.






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