Over the past decade, analysis of whole-genome expression profiling with DNA microarrays has revolutionized the way biologists study gene function. RNA amplification has become the preferred method of preparing probes for most microarray platforms. Unfortunately, the benefits of RNA amplification have not been easily accessible to researchers analyzing bacterial gene expression profiles. The absence of poly(A) tails on bacterial mRNA prevents the use of oligo(dT) primed reverse transcription, which is at the heart of most amplification methods. The lack of an appropriate method for bacterial RNA amplification has hampered studies that rely on rare or limited samples, such as environmental collections, small volume cultures, and bacterial RNA isolated from host cells. With over 160 microbial genomes fully sequenced, and many more in progress, an unprecedented opportunity to perform global expression analysis on important pathogens and model organisms is now available.
MessageAmp II-Bacteria Kit
MessageAmp II-Bacteria (patent pending) is based on a linear RNA amplification technology that enables whole genome expression analysis from limited bacterial samples. By amplifying RNA, microarray analyses can now be performed with up to 500-fold less starting material. MessageAmp II-Bacteria can be used with any bacterial species and is compatible with purified total RNA, bacterial mRNA enriched using Ambions MICROBExpress Kit (see sidebar), or bacterial RNA enriched from host cell mixtures with the MICROBEnrich Kit (see sidebar). As little as 10 ng of enriched bacterial mRNA or 100 ng of total RNA can be amplified to yield sufficient antisense amplified RNA (aRNA) for microarray analysis.
Linear Bacterial RNA Amplification
MessageAmp II-Bacteria is based on a linear amplification method (Figure 1) in which the RNA sample is first polyadenylated with Poly(A) Polymerase, then reverse transcribed using oligo(dT) primers containing a T7 RNA polymerase promoter. The resulting cDNA is used to produce aRNA by in vitro transcription (IVT) primed by the added T7 promoter. The aRNA can be labeled during IVT with a variety of modified nucleotides (for glass slide microarrays or Affymetrix GeneChip arrays), or it can be used to generate 33P-labeled sense strand cDNA (for nylon PCR arrays).
Figure 1. Schematic of MessageAmp II-Bacteria Procedure.
Yields of Amplified RNA
It is important to understand that the amplified RNA yields obtained with the MessageAmp II-Bacteria Kit or any amplification system will vary based on factors such as RNA isolation method, input RNA amount, RNA quality, the bacterial species from which the RNA was isolated, and incubation time of the in vitro transc ription reaction. Total RNA isolation methods that include a glass fiber filter based purification step, such as Ambions RiboPure-Bacteria Kit, give higher aRNA yields than organic extraction methods. RNA purity and intactness can also have a significant impact on amplification. Figure 2 shows typical aRNA yields for 5 bacterial species using 100 ng of total RNA isolated with the RiboPure-Bacteria Kit and amplified using MessageAmp II-Bacteria and a 6 hour IVT reaction. Even with a short 6 hour IVT incubation time, 100 ng of input total RNA results in sufficient aRNA for several replicate array experiments. A 14 hour IVT incubation is recommended when <100 ng of input RNA is used.
Figure 2. MessageAmp II-Bacteria aRNA Yields. MessageAmp II-Bacteria yields of aRNA (g) for 5 different bacterial species. Yields are averages from triplicate, 6 hour in vitro transcription reactions using 100 ng total RNA. E. coli was tested with both 100 ng and 10 ng total RNA input. aRNA concentrations were measured by UV absorbance.
Whole Genome Microarray Analysis
Affymetrix E. coli Antisense Whole Genome Arrays were used to compare biotinylated MessageAmp II-Bacteria aRNA derived from E. coli total RNA and MICROBExpress enriched mRNA, as well as the recommended Affymetrix labeling protocol using 10 g E. coli total RNA. The replicate GeneChip arrays shown in Figure 3 demonstrate high reproducibility for the MessageAmp II-Bacteria technology and the Affymetrix recommended labeling protocol. Pearsons Correlation Coefficients for all technical replicate comparisons was >0.995. The sensitivity (% Present Calls) obtained with MessageAmp II-Bacteria is comparable to the standard Affymetrix labeling protocol, as is the call concordance between replicates. (See article in previous issue for a more complete definition of sensitivity and concordance analysis.) Thus, MessageAmp II-Bacteria enables you to use significantly less starting material without compromising the quality of microarray data.
Figure 3. Affymetrix E. coli Antisense Genome Array Replicate Analysis. Scatter plots of log signal for replicate labeling reactions using Affymetrix E. coli antisense whole genome arrays (all 7312 probe sets are shown). Either E. coli total RNA or enriched mRNA derived from it (MICROBExpress Kit) was used with the recommended Affymetrix labeling protocol or Ambions MessageAmp II-Bacteria biotin IVT labeling protocol. The data was analyzed with Affymetrix Microarray Suite, Version 5 with scaling to all probe sets and a target signal value of 500. 2, 3, 10 and 30-fold change lines are represented.
Tips for Bacterial RNA Amplification
When using MessageAmp II-Bacteria for expression analysis, Ambion recommends that the researcher use similar input RNA mass amounts, RNA type (total RNA or mRNA), and IVT incubation times when performing comparisons between samples. Also, as with other RNA amplification methods, only amplified samples should be compared to one another. Unamplified samples should not be compared directly with amplified samples. All amplification methods, or any labeling method for that matter, produce some biases in mRNA representation. What is important for expression analysis is that the biases are reproducible within a method and that similar labeling methods are used on samples being compared. MessageAmp II-Bacteria compares favorably with standard labeling protocols in terms of reproducibility and dynamic range, but with a dramatically reduced requirement for input RNA.
A Complete Kit for Bacterial RNA Amplification
The MessageAmp II-Bacteria Kit provides reagents for 20 RNA amplifications starting with as little as 10 ng bacterial mRNA or as much as 500 ng total RNA. A variety of labeling methods can be used with the kit, including 33P cDNA labeling, direct labeling with Cy dyes or biotin, and indirect coupling using aminoallyl nucleotides (modified nucleotides are not provided with the kit). E. coli total RNA is provided as a control.
While MessageAmp II-Bacteria can be successfully used with total RNA, MICROBExpress enriched bacterial mRNA is a much better choice for microarray analysis. A 10-fold to 100-fold increase in sensitivity is seen when using enriched mRNA, compared with total RNA. Overall signal and signal-to-noise ratios can be dramatically increased by using RNA prepared using the MICROBEnrich and/or MICROBExpress Kits with MessageAmp II-Bacteria .
Cat# Product Name Size 1790 MessageAmp II-Bacteria RNA Amplification Kit 20 rxns 1901 MICROBEnrich Kit 20 purifications 1905 MICROBExpress Bacterial mRNA Enrichment Kit 20 rxns 1925 RiboPure-Bacteria Kit 50 purifications