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.
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
There are several expression analysis techniques that are adaptable to multi-target analysis, including Northerns, RT-PCR, and nuclease protection assays (NPAs). Each of these techniques has advantages when studying gene expression; Northerns reveal transcript size and splice variants, RT-PCR has the greatest sensitivity, and NPAs are the easiest method to utilize multi-target analysis. In order to measure the level of more than one RNA with Northerns, it is typically necessary to go through a cyclical process of probing, autoradiography, stripping and reprobing the blot with other probes. In addition to being time-consuming, this process can result in inconsistency due to loss of target RNA and incomplete stripping of the probe. RT-PCR is typically limited to coamplification of one or two targets, since unexpected products often result as the number of primer pairs increases. Additionally, optimization of multi-target RT-PCR, including necessary pilot experiments to determine exponential phase PCR conditions for each message under study, is often a very time consuming process. In contrast, a single NPA assay can accommodate five to ten probes plus one or two internal controls, allowing simultaneous quantitation of multiple messages within a single RNA sample. And, unlike Northern analysis, multiple probes may be added to one hybridization reaction allowing multiple targets to be assessed simultaneously
Multi-Probe Considerations
How Many Probes Can Be Used at Once?
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.
Figure 1. Simultaneous Quantitation of Multiple mRNAs Using RPA III. Ten micrograms of various mouse tissue total RNAs were hybridized overnight with approximately 50,000 cpm each of seven distinct probe transcripts. Nuclease digestion, product separation on a denaturing 6% acrylamide gel, and a four hour exposure to film at -80°C were used to assess hybridization levels. The radiolabeled probes were synthesized in 5 µl transcription reactions using Ambin's MAXIscript Kit with [alpha-32P]UTP (800 Ci/mmol, 10 mCi/ml) and gel purified prior to hybridization. The specific activities of the cyclophilin and ß-actin probes were reduced twenty and 200-fold, respectively, by adding appropriate amounts of nonradioactive UTP to the transcription reaction.
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
One of the most common mistakes in designing a nuclease protection experiment is the addition of too much probe. This issue is further complicated when performing multi-target analysis where the abundance of the messages under study may vary widely (i.e. a rare message and an abundant internal control message are being measured simultaneously). NPAs require just enough probe to be in molar excess of the target mRNA. Therefore, abundant messages require a greater mass amount of probe than rare messages to achieve molar excess. If the probes are labeled to the same specific activity, the abundant message will have many more labeled rNTPs present in the final product, or protected fragment, than the rare message. This can be problematic in the final autoradiography, as the signal from the internal control may be so strong that it interferes with the visualization of the signal from the rare message. Additionally, to be quantitative, the signal from the internal control must be in the linear range of the film. A strong signal requires a short exposure time to be in linear range, making simultaneous visualization of the signal from the rare message very difficult.
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.
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.
Sensitive andFlexible NPA Analysis
The nuclease protection assay is an extremely sensitive technique for the detection, quantitation, and characterization of RNA. While the exquisite sensitivity and convenience of Ambion's single-tube, Proteinase K-free and phenol-free kits have made them a favorite among researchers studying RNA expression, perhaps the single greatest benefit of nuclease protection assays is their compatibility with multi-target analysis. While all of Ambion's NPA kits work well in a multi-probe format, for analysis of rare messages where sensitivity is the most important consideration, choose the RPA III Kit. It is the most sensitive NPA kit available and is capable of analyzing up to 10 RNA probes in a single tube.