A Sensitive Method of Nonisotopic Detection that Rivals 32P Labeled Probes
Nonisotopic probes have typically been made by enzymatic incorporation of modified nucleotides into RNA transcripts. Now, the BrightStar™ Psoralen-Biotin Kit provides a method to label any nucleic acid, post-synthesis, with biotin via a chemical reaction, which covalently bonds the label to the nucleic acid. At Ambion, we have found these probes to be more sensitive than those made be enzymatic incorporation. The BrightStar Psoralen-Biotin Kit is part of Ambion's BrightStar System, which includes BrightStar™-Plus Positively Charged Membranes, the BrightStar™ Psoralen-Biotin Probe Labeling Kit, and the BrightStar™ BioDetectà Nonisotopic Detection Kit. This system is compatible with all of Ambion's Northern analysis products. |
Sensitivity
At Ambion, we find Psoralen-Biotin labeling to be two to four times more sensitive than enzymatic labeling using biotinylated nucleotides. We also find that if a 32P signal can be visualized in three days or less when exposed at -80åC with one intensifying screen, the BrightStar System will give equivalent sensitivity, and will often generate signal in exposures of one hour or less. Because of the nature of the nonisotopic substrate, excessively long exposures (greater than 18 hours) can result in increased background, obscuring the signal of interest. Therefore, exposures of greater than two hours are rarely made. However, this is not the case with 32P experiments. An isotopic blot can often be exposed for a week or more. We have seen a 32P-labeled RNA probe detect as little as 4 fg of artificial sense strand in an RPA exposed seven days with an intensifying screen. For most analyses, the BrightStar system has sufficient sensitivity to be an excellent alternative to isotopic detection. When necessary, additional sensitivity can be achieved by using more sample RNA in the assay (up to 80 - 100 µg in an RPA or 20 µg in a Northern) or switching to poly(A) RNA. However, if the message of interest is extremely rare, isotopic systems may be the only suitable method of detection.
Data
In this experiment, triplicate Northern blots were prepared using Ambion's NorthernMaxà Kit. Each blot contained a titration of mouse liver total RNA ranging from 10 µg down to 80 ng. High specific activity RNA probes complementary to GAPDH message were generated using the MAXIscript™ Kit and each of the three labeling methods. 10 ng probe/ml NorthernMax hybridization solution was used in both nonisotopic hybridizations, while 1 x 106 cpm probe/ml NorthernMax hybridization solution was used in the isotopic hybridization. The blots were hybridized overnight at 68°C, and post hybridization washes were performed according to Ambion's NorthernMax Kit procedure. The nonisotopic probes were detected simultaneously using the BioDetect Kit and exposed to the same piece of film. A 30-minute exposure at peak light emission of the Psoralen-Biotin blot gave equivalent signal to the 32P blot exposed to film for 20 hours at -80°C with one intensifying screen. In this Northern analysis, Psoralen-Biotin labeled RNA probes were approximately two times more sensitive than those labeled enzymatically using biotinylated nucleotides, and are equivalent to those labeled with 32P. Both detected GAPDH mRNA in 80 ng total RNA; however, the enzymatically labeled biotin probe required 160 ng of total RNA to detect the message (exposed for 30 minutes at peak light emission).
Figure 1. Sensitivity of Psoralen-Biotin, Enzymatic Biotin and 32P-labeled Probes in a Northern. Triplicate blots were prepared using Ambion's NorthernMax Kit and dilutions of mouse liver total RNA. Blots were hybridized with GAPDH probes labeled with Psoralen-Biotin, biotin incorporated enzymatically, or 32P. Panel A blots were hybridized with antisense RNA probes at 68°C overnight while Panel B blots were hybridized with DNA probes at 42°C.
A similar comparison was made using DNA probes (Panel B). Again, triplicate blots were prepared using NorthernMax and a titration of total RNA from 5 µg to 50 ng. GAPDH DNA probes were generated by
10 ng probe/ml NorthernMax hybridization solution were used in the nonisotopic hybridization, and 1 x 106 cpm probe/ml NorthernMax hybridization solution were used for the isotopic probe. The blots were hybridized at 42°C overnight and washed according to the NorthernMax protocol. The nonisotopic probes were detected simultaneously using the BioDetect Kit and exposed to the same piece of film.
The Psoralen-Biotin labeled probe detected GAPDH message in 200 ng of RNA, whereas the enzymatically labeled biotin probe required 500 ng of RNA to achieve similar signal in a two hour exposure at peak light emission. When compared to the blot hybridized with the 32P-labeled probe (exposed for 48 hours with one intensifying screen), the Psoralen-Biotin blot gave equivalent or greater signal in 200 ng of total RNA. We found using DNA probes in Northerns, that Psoralen-Biotin labeled probes show approximately two times more sensitivity than enzymatically labeled probes, and equal or greater sensitivity when compared to 32P-labeled probes.
Figure 2. Sensitivity of Psoralen-Biotin vs. 32P-labeled Probe in a Northern. Duplicate Northern blots were prepared using the NorthernMax Kit. Antisense RNA probes to the GAPDH message were labeled with Psoralen-Biotin or 32P and hybridized overnight at 68°C. Blots were washed and the 32P blot was exposed to film, while the Psoralen-Biotin blot was detected and exposed to film.
In a second Northern blot experiment, Psoralen-Biotin and 32P-labeled RNA probes were hybridized to total RNA ranging in concentration from 150 ng to 2.5 ng (Figure 2). Duplicate blots were prepared using the NorthernMax Kit, and hybridized with an RNA probe complementary to GAPDH message under the same conditions as in Figure 2, described above. Both methods detected GAPDH message in 5 ng of total RNA. The 32P blot required an exposure of four days at -80åC with one intensifying screen, while the Psoralen-Biotin blot revealed similar or greater signal when exposed to film for only one hour at peak light emission.
A similar comparison was made using DNA probes (Panel B). Again, triplicate blots were prepared using NorthernMax and a titration of total RNA from 5 µg to 50 ng. GAPDH DNA probes were generated by
- random-priming (i.e. enzymatic) reactions incorporating biotin-14-dCTP, or
- 32P-dATP, and by
- direct labeling of the DNA template with Psoralen-Biotin.
10 ng probe/ml NorthernMax hybridization solution were used in the nonisotopic hybridization, and 1 x 106 cpm probe/ml NorthernMax hybridization solution were used for the isotopic probe. The blots were hybridized at 42°C overnight and washed according to the NorthernMax protocol. The nonisotopic probes were detected simultaneously using the BioDetect Kit and exposed to the same piece of film.
The Psoralen-Biotin labeled probe detected GAPDH message in 200 ng of RNA, whereas the enzymatically labeled biotin probe required 500 ng of RNA to achieve similar signal in a two hour exposure at peak light emission. When compared to the blot hybridized with the 32P-labeled probe (exposed for 48 hours with one intensifying screen), the Psoralen-Biotin blot gave equivalent or greater signal in 200 ng of total RNA. We found using DNA probes in Northerns, that Psoralen-Biotin labeled probes show approximately two times more sensitivity than enzymatically labeled probes, and equal or greater sensitivity when compared to 32P-labeled probes.
Dot blots (data not shown)
Dilutions of artificial sense strand RNA were hybridized with antisense RNA labeled with either
Triplicate blots were prepared using dilutions of in vitro transcribed -actin sense strand. The different mass amounts of sense RNA were adjusted to the same final mass using yeast RNA, and then spotted onto the membranes. Identical antisense RNA transcripts were prepared by in vitro transcription using Ambion's MAXIscript Kit, and were labeled by each of the three methods. The blots were hybridized using the recommended amounts of probes (same as in Figure 1) at 68°C overnight and washed according to the NorthernMax protocol. The nonisotopic blots were detected simultaneously using the BrightStar BioDetect Kit and exposed to the same piece of film (Kodak XAR). The nonisotopic blots were exposed to film for 30 minutes during peak light emission of the substrate, CDP-StarÃ. The blot hybridized with a 32P-labeled probe was exposed for four days at -80°C with one intensifying screen. All three methods detected 100 fg of artificial sense strand in the exposure times as described. The data presented here demonstrate that Ambion's BrightStar system for nonisotopic labeling and detection of nucleic acids is a viable alternative to using isotopically labeled probes. Furthermore, probes generated using the Psoralen-Biotin labeling kit are as sensitive, if not more, than probes biotinylated using the more common method of enzymatic incorporation.
RPAs
While probes labeled post-synthetically with Psoralen-Biotin work very robustly in blot hybridizations, results with such probes in nuclease protection assays are at best inconsistent. We therefore recommend that probes used in ribonuclease protection assays be labeled using enzymatically incorporated, nonisotopically modified nucleotides.
- Psoralen Biotin
- Biotin-14-CTP or
- 32P-UTP.
Triplicate blots were prepared using dilutions of in vitro transcribed -actin sense strand. The different mass amounts of sense RNA were adjusted to the same final mass using yeast RNA, and then spotted onto the membranes. Identical antisense RNA transcripts were prepared by in vitro transcription using Ambion's MAXIscript Kit, and were labeled by each of the three methods. The blots were hybridized using the recommended amounts of probes (same as in Figure 1) at 68°C overnight and washed according to the NorthernMax protocol. The nonisotopic blots were detected simultaneously using the BrightStar BioDetect Kit and exposed to the same piece of film (Kodak XAR). The nonisotopic blots were exposed to film for 30 minutes during peak light emission of the substrate, CDP-StarÃ. The blot hybridized with a 32P-labeled probe was exposed for four days at -80°C with one intensifying screen. All three methods detected 100 fg of artificial sense strand in the exposure times as described. The data presented here demonstrate that Ambion's BrightStar system for nonisotopic labeling and detection of nucleic acids is a viable alternative to using isotopically labeled probes. Furthermore, probes generated using the Psoralen-Biotin labeling kit are as sensitive, if not more, than probes biotinylated using the more common method of enzymatic incorporation.
RPAs
While probes labeled post-synthetically with Psoralen-Biotin work very robustly in blot hybridizations, results with such probes in nuclease protection assays are at best inconsistent. We therefore recommend that probes used in ribonuclease protection assays be labeled using enzymatically incorporated, nonisotopically modified nucleotides.
