Your Data: Comparison of Two Priming Strategies for RNA Amplification

Mycobacterium tuberculosis, the primary cause of tuberculosis (TB), kills more people worldwide every year than any other bacterial pathogen. Gene expression profiling of M. tuberculosis provides insight into host-pathogen interactions and has the potential to identify therapeutic targets for more effective TB treatment. Dr. Simon Waddell and colleagues compared the Ambion® MessageAmp™ II-Bacteria RNA Amplification Kit with a second priming strategy for first strand synthesis in the production of amplified RNA (aRNA) from Mycobacterium tuberculosis [1]. In microarray experiments, the MessageAmp method resulted in greater aRNA yield, increased aRNA size distribution, and decreased mRNA representation bias than the second method.

Experimental Approach
The strategy employed in the MessageAmp™ II- Bacteria RNA Amplification Kit is to first polyadenylate bacterial RNA and then amplify using a T7 oligo(dT) primer and T7 RNA Polymerase. In the first of three publications using this kit, Dr. Simon Waddell and colleagues evaluated two different priming strategies for first strand synthesis in the production of aRNA from Mycobacterium tuberculosis [1]. The MessageAmp II-Bacteria Kit was compared to a second system using a set of mycobacterial amplification directed primers (ADP). These primers were designed to prime all genes annotated in the M. tuberculosis genome.

Size distribution, aRNA yield, reproducibility, and representational bias were compared as were the gene expression profiles of unamplified to amplified RNA in a well-characterized system.

Results
Compared to the ADP priming strategy, the MessageAmp II-Bacteria RNA Amplification Kit was able to produce sufficient yields of aRNA for microarray hybridization with lower RNA input amounts (5 ng compared with 50 ng input needed for the ADP system), and produced aRNA with a larger modal size.

aRNA yields and size distribution were highly reproducible between both methods of amplification. In addition, when replicate samples were amplified and hybridized either on the same or different days, correlation coefficients of the ranked signal of all genes were high for both amplification methods.

When compared to the expression profiles from unamplified RNA, aRNA from both priming strategies showed that some genes were over- or under-represented. This finding confirms the need to use the same amplification strategy on all samples that will be compared.

The authors also looked at the extent to which some genes in the amplified RNA were not detected by microarray analysis compared to genes detected using unamplified RNA. Though both systems resulted in the loss of detection of some genes, the ADP system resulted in a higher number of undetected genes. This problem was exacerbated when input amounts were lowered to 5 ng. It was noted however, that the majority of genes not detected by microarray analysis using amplified RNA, overlapped with those determined to be under the detection threshold using unamplified RNA.

Finally, the authors used two well-characterized M. tuberculosis growth systems (aerobic vs. non-replicating persistence, NRP1) to measure the overall effectiveness of amplified bacterial RNA in detecting differential gene expression. Data from amplified RNA was compared to data from unamplified RNA. The MessageAmp amplification method correlated most closely with the expression profiles identified using unamplified RNA (Figure 1).

Figure 1. Gene Expression Profiles from the MessageAmp™ Strategy More Closely Correlate with that of Unamplified RNA. A Spearman’s rank correlation of the 155 genes identified whose expression was significantly differentially expressed in microaerophilic compared to aerobic M. tuberculosis growth conditions using unamplified RNA (marked Un). The mean gene expression ratios derived from unamplified RNA and from the products of 500, 50, and 5 ng amplifications using oligo-dT (MessageAmp™) and ADP methods are displayed. Genes are ordered in rows, amplification conditions as columns. Red coloring indicates genes induced in microaerophilic vs. aerobic growth conditions; green coloring denotes repression.

Subsequent Studies
Waddell and colleagues went on to use the MessageAmp II-Bacteria RNA Amplification Kit to study M. tuberculosis in at least two subsequent publications [2, 3]. Whole genome microarrays were used to gain insight into host-pathogen interactions by simultaneously studying transcriptional changes in M. tuberculosis and in its main host cells, macrophages and dendritic cells.

In a third publication, the authors identified a persister-like bacilli in the sputum of patients infected with tuberculosis.

These studies will pave the way for further understanding of M. tuberculosis infection, spread, and host cell interactions—with the ultimate goal of developing effective treatments and preventative measures for tuberculosis.

Data used with permission.