Invitrogen™ ULTRAhyb™ Ultrasensitive Hybridization Buffer

The extreme sensitivity of ULTRAhyb Ultrasensitive Hybridization Buffer may detect RNAs that are not the expected full-length target. Although much of the probe binding can be legitimate (hybridization to alternatively spliced, partially degraded, or closely related mRNAs), some might be hybridization to RNAs with only partial homology to the target. We recommend using a more specific probe if possible. Here are other possible causes for cross-hybridization and solutions offered:
- The hybridization stringency may have been inadequate. Increasing the hybridization and wash temperature 3-10 degrees C can greatly reduce the levels of non-target hybridization. Simply reducing the amount of time used to expose the blot to film might also alleviate the problem.

- The probes might have contained non-target sequence. The presence of vector sequence within the probe can cause hybridization to RNAs sharing sequence homology with the vector. If the probe template contains vector sequence, cleave it by restriction digestion and then gel purify the sequence of interest before labeling.

- Too much non-isotopic probe may have been present. Non-isotopic probes can have problems with cross-hybridization, especially when they are used at 42 degrees C. We have observed that lowering the probe concentration 10 to 100-fold in the hybridization reaction will greatly reduce non-specific hybridization while having little if any impact on target-specific hybridization.

Here are possible causes and solutions:
- Not enough probe (or label) was used. Using less than the recommended amounts of probe, using low specific activity probe, or using less than full-length probes can lead to low signal. We recommend checking each of these factors if low sensitivity is observed.

- The hybridization and washes may have been too stringent. We recommend lowering the hybridization temperature or the wash time and temperature. This may be especially helpful for oligonucleotide probes. Note that reducing stringency can lead to higher background and cross-hybridization.

Here are possible causes and solutions:
- There could be precipitates in the ULTRAhyb Ultrasensitive Hybridization Buffer. Inadequate solubilization of the hybridization buffer is one of the primary causes of high background. We recommend increasing the amount of time used to preheat the buffer and ensure that there is no precipitate in the buffer before adding it to the blot. ULTRAhyb Ultrasensitive Hybridization Buffer may start to precipitate at temperatures below 25-30 degrees C.

- Prehybridization may have been inadequate. We recommend increasing the blot prehybridization time from 30 min to 1 hr.

- The probe may have been too old. We recommend using fresh probe. Isotopic probes that are several days old tend to produce higher background than freshly prepared probes. This is attributed to probe size; radiolytic decay reduces the size of the probe molecules over time.

- Unincorporated radionucleotides may have been present. Although we don't ordinarily recommend removing free nucleotides, we have occasionally observed high background from unincorporated label. We recommend removing free label by precipitation (0.5 M NH4OAc and 2 volumes EtOH) or with a spin column designed for this purpose.

- Hybridization stringency may have been inadequate. We recommend the following:
-If hybridizing at 42 degrees C, try raising the hybridization temperature to 4 degrees C, or even to 55 degrees C.
-If hybridizing at 68 degrees C, hybridization stringency is unlikely to be causing background. In our experience, raising the hybridization temperature above 68 degrees C does not decrease background.

- Ionic interactions could have led to high background. If the background signal makes the blot look uniformly dark, we recommned adding a high salt wash to minimize ionic interactions between the probe and the hybridization membrane. To do this, after the ordinary washes, add a 2 x 15 min wash in 5X SSC or SSPE, 0.5% SDS at 68 degrees C for RNA probes, or at 60 degrees C for DNA probes.

- Washing may have been inadequate. We recommend the following:
-Double check that your wash buffers contain SDS. Wash buffers lacking SDS are not recommended for use with ULTRAhyb Ultrasensitive Hybridization Buffer. Doubling the wash times and/or washing at higher temperatures can reduce background. Wash temperatures can be raised from 42 degrees C to 55 degrees C or 60 degrees C.
-If you are washing at 68 degrees C, inadequate washing is probably not causing your high background. In our experience, raising wash temperatures above 68 degrees C does not decrease background.