Protocol Online logo
Top : Forum Archives: : Molecular Biology

Both total RNA and mRNA in the same reaction? - (Apr/23/2005 )

I wonder why some protocols add both total RNA and mRNA in the same reaction. What for? rolleyes.gif For example this one:

cDNA Synthesis:

Mix together in PCR RNase free tube (Ambion#12225):
1 ng total RNA
0.1 ng Rubisco RCA mRNA.

Add nuclease-free water (ex. Gibco #10977) to bring the volume to 13 l
1 ul of anchored oligo dT primer (500 ng/ul stock conc.). The final [oligo-dT] will be 0.5 mM in 20ul volume.



Dear friend,
Me again : )
Is the Rubisco RCA mRNA provided by Ambion kit?
If yes, It served as internal control. Meaning that you should at least get cDNA for Rubisco RCA if your target gene is not being amplify. If you can't get cDNA for both, that mean your RT step was failed.

Regards : )