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Normalizing RNA concentrations prior to cDNA production


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#1 phosphate girl

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Posted 12 April 2010 - 05:46 AM

Hello,

Can anyone out there give me some advice please I am looking at the expression of a gene in plants grown under different conditions. The plan is to extract RNA, make cDNA and then run qPCR and compare expression between samples.

In order to do this I need to make sure I have the same concentration of cDNA in all my samples correct? I can do this by making sure I start off with similar concens of extracted RNA across samples. I am a bit of a novice - is the best way to do this with a nanodrop to determine RNA concentrations and then dilute with pure water/buffer to get samples all in the same range and then carry out cDNA synthesis?

Any other hints and tips for getting good quality cDNA for qPCR would be much appreciated for a first timer! Fingers crossed it works

#2 BryanC

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Posted 12 April 2010 - 06:06 AM

Do spec your RNA, and a nanodrop works great. However, I would not dilute the entire tube of the RNA.

In your cDNA synthesis kit, there will be a recommended starting amount of RNA.(eg VILO kit from invitrogen is up to 2.5ug RNA per 20 ul rxn) Keeping the VILO kit as an example.

Its recommended recipe per rxn is:
4ul Buffer
2ul Enzyme
X ul of RNA
Bring to 20ul with depc H20.
Total vol is 20ul

This is where you normalize your starting RNA. Find the the conc for your RNA samples. Calculate the volume for each that will equal the starting amount you will be using, then bring that volume up to 14ul with H20. Now add your buffer, then enzyme(I make a master mix for this step then aliquot it to my normalized RNA samples).

Hello,

Can anyone out there give me some advice please I am looking at the expression of a gene in plants grown under different conditions. The plan is to extract RNA, make cDNA and then run qPCR and compare expression between samples.

In order to do this I need to make sure I have the same concentration of cDNA in all my samples correct? I can do this by making sure I start off with similar concens of extracted RNA across samples. I am a bit of a novice - is the best way to do this with a nanodrop to determine RNA concentrations and then dilute with pure water/buffer to get samples all in the same range and then carry out cDNA synthesis?

Any other hints and tips for getting good quality cDNA for qPCR would be much appreciated for a first timer! Fingers crossed it works


Edited by BryanC, 12 April 2010 - 06:07 AM.





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