PCR Products from Reverse Transcribed RNA without RT-primers:

I would like you to feature on the website under “tech corner” this important technical information that we discovered in our lab (Professor Kenneth M. Baldwin at the University of California Irvine) in the analyses of sense and naturally occurring antisense RNA products.

Reverse transcription makes cDNA of the RNA and is usually primed by an antisense primer (figure 1A and 1B). The generated cDNA is amplified using PCR primers targeting a region of the cDNA. A negative control for such a reaction is normally a non reverse transcribed RNA. We normally have all the ingredient of the RT-reaction except the reverse transcriptase, and we use this as the negative control for the PCR reaction to compare with reverse transcribed RNA. We always obtain a negative signal for the PCR reaction on a non-reverse transcribed DNase treated RNA insuring us of the effectiveness of the DNase treatment and of the cDNA-specificity of the signal. As additional negative control for the PCR reactions we decided to run the reverse transcription reaction without RT primers. To our surprise, the PCR reaction gave a strong signal that was equivalent in strength to 5 - 25% of the total signal obtained when we run the reaction with specific RT primers. We observed that the intensity of the no primer RT-generated signal varied depending on the location of the PCR primers along the gene of interest, as well as with the experimental manipulation. Genrally, the intensity of the signal covaried with the abundance of the antisense RNA. Based on this observation we decided to run no primer RT for all PCR reactions and treat the obtained signal as a background to be subtracted from the specifically primed RT-PCR reaction signal. If one was to study the regulation and abundance of a transcript that also may have an antisense component, one should always carry two RT reactions of the same RNA: a no primer RT and the antisense specific primer RT. These reactions should be used for the PCR amplification using the same PCR primers and same dilutions and cycling conditions. The assessment for the signal should be based on the difference between the primed RT reaction and the non-primed RT reaction PCR signals. As a standard, for all RT-PCR analyses of RNA on a locus which has both strands transcribed, a background signal coming from a non-primed RT-PCR reaction signal should always be subtracted from the primed RT-PCR signal.

Note that in our hands in analyzing the cardiac MHC RNAs, the non primed RT-signal was much more evident in PCR targeting the primary transcript (pre-mRNA). As for the processed mRNA, the non-primed RT-PCR signal was very negligent in intensity (<1% of the specifically primed RT) that could be ignored without major influence on the interpretation of the results.  We have not yet reported this important information to the science world yet, but we are in the process of writing a manuscript that will have this phenomenon reported. (update will follow).

Figure 1 below illustrates a schematic of the RT-PCR reactions of the sense and antisense RNA of the gene locus: (A) the RT target is the sense RNA strand using an antisense RT-primer. The reverse transcriptase makes a complement of the RNA (cDNA) which is subequently amplified with the PCR primer pair. (B) the RT target is the antisense RNA using a sense RT-primer. The reverse transcriptase makes a complement of the RNA (cDNA) which is subsequently amplified with the PCR primer set. (C) to explain the PCR signal generated from non-primed RT reaction, we propose that the RNA hybridize to its antisense and prime the reaction so that the reverse transcription work to fill in the 3’ overhang. It is assumed that the same fill in reaction also occurs in each of the specifically primed RT-reactions.