Simultaneous Detection of Multiple mRNA Targets
|Advantages of Multi-target Analysis |
For most laboratories studying gene expression, it is rarely sufficient to assess the expression level of a single gene. Multi-target analysis increases the quantity of gene expression data that can be gathered in a single assay, and it increases the quality of data obtained compared to analyzing multiple mRNAs individually. Experimental variability is reduced or eliminated by measuring levels of multiple target mRNAs in the same sample. These analyses typically include an internal control RNA, such as glyceraldehyde-3-phoshate dehydrogenase (GAPDH), ß-actin, or ribosomal RNA, the level of which remains constant across the sample RNAs being studied.
NPAs Offer Advantages Over Other Techniques
While it is possible to use ten to twelve probes simultaneously in an NPA, five or six is probably a more reasonable number. Figure 1 demonstrates the use of Ambion's RPA III™ Kit for multiple probe analysis of five oncogenes and two internal controls, all within individual total RNA samples from several mouse tissues. When performing multi-probe NPAs, it is important that each probe differ in size and that each give a single band when used in an assay alone. Effective separation of all 7 probes seen in Figure 1 was achieved on a 6% denaturing polyacrylamide gel. Typically, a 5-6% denaturing polyacrylamide gel provides efficient separation of probes ranging from 100-500 bases in length. Higher percentages of acrylamide should be used for shorter probes.
Simultaneous Detection of Rare and Abundant Messages
Lowering the specific activity (cpm or amount of label per microgram) of probe used for more abundant messages easily solves the problem of achieving similar signal intensities. Moderately abundant mRNAs (like the common internal controls ß-actin and cyclophilin) make up approximately 0.1% of total RNA, whereas rare mRNAs make up <0.001%. Therefore, reducing probe specific activity between 20 and 200 fold, depending on true differences in abundance and the length of the probe (shorter probes are less sensitive), is typically enough to achieve comparable signal intensities. Ribosomal RNAs make up approximately 80% of total RNA and therefore need to be labeled to an extremely low specific activity. Figure 1 shows that by reducing the specific activities by 20 and 200 fold for the internal control probes to cyclophilin and ß-actin, respectively, it was possible to simultaneously detect these moderately abundant messages with such rare messages as c-myc and Jun B. Probe specific activity was decreased by adding appropriate amounts of unlabeled UTP to the probe synthesis reaction. By lowering the specific activities of probes for abundant messages, larger mass amounts of probe may be added to the assay to achieve the molar excess necessary for complete hybridization without interfering with the visualization of the rare message.