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™ BioDetect™ Nonisotopic Detection Kit. This system is compatible with all of Ambion's Northern analysis products.
In this experiment, triplicate Northern blots were prepared using Ambion's NorthernMax® 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).
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.
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.
Dot blots (data not shown)
Dilutions of artificial sense strand RNA were hybridized with antisense RNA labeled with either:
- Psoralen Biotin
- Biotin-14-CTP or
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-Star®. 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.
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.