I use b-actin as my housekeeping and I don´t have that problem, and the technical replicates (pippeting) of the 47s rRNA work fine as well, so...I am about to try with the third pair of primers, but I am afraid there´s something about the biology of the rRNA that I am losing. Thanks
The primers needs to be optimized for equal efficiency of amplification using 4-5 diluted template dilutions and a graph needs to plotted accordingly before performing the test reaction. please see the Fig 1. of the article:
http://www.gene-quantification.net/livak-2001.pdf
rRNA genes occur in tandem repeats and it may pose amplification errors in replicates due to miscalculated optimization caused by high quantity of available template.
Secondly, rRNA lacks the PolyA and the cDNA needs to be prepared using random nonamers or hexamers which can lead to up to 20 fold erroneous amplification errors. This may result the technical replicate values to fall close but biological replicates to vary significantly.
Does the third primer behave well with cDNA prepared from rRNA specific primer? Furthermore, using two or more sets of internal reference may be valuable to predict biological causes for the variation observed.
Last point is how are the melt curves? Many times when the primer is not able to amplify from biological replicates either due to variation in quality of RNA, biological cause or sheer probability they may form primer dimers that add up fluorescent counts.
To avoid, differences between technical replicates it is best to use low concentration of stocks (higher volumes) and adjust the calculations by reducing the final water volume. It works many times.
Thanks and best of luck
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