Northern analysis remains a standard method for the detection and quantitation of mRNA despite the advent of more sensitive techniques such as RT-PCR. In addition to being a quantitative tool, Northern analysis has the advantage of being the only method able to simultaneously provide information about the size, alternative splicing and integrity of RNA samples. However, inefficiencies in any of the several steps in the procedure, or ribonuclease contamination can lead to a loss in the sensitivity of Northern analysis. Ambion's Northern Max™ Kits and reagents eliminate these procedural inefficiencies, and are designed to maximize sensitivity.
Complete Northern Systems
The NorthernMax™ Kits
are complete systems for Northern blotting that combine ultrasensitive reagents and reliable protocols with unsurpassed quality control, ensuring optimal results in less time. The NorthernMax Kits include ULTRAhyb™ Ultrasensitive Hybridization Buffer
, which can increase sensitivity up to 100X compared to other hybridization solutions. Figure 1 (below) demonstrates the increase in sensitivity observed with ULTRAhyb compared to 2 standard hybridization solutions. Because ULTRAhyb maximizes blot sensitivity, hybridization can be completed in just 2 hr, and film exposure times may also be significantly shortened for many messages. The NorthernMax procedure also incorporates a rapid, alkaline transfer procedure that increases sensitivity by efficiently moving RNA, especially larger transcripts, onto the membrane. This step takes only 2 hr, and it can trim almost an entire day off the standard procedure, which generally requires an overnight transfer. By saving time on both the RNA transfer and probe hybridization steps, the entire NorthernMax procedure can be completed in just 1 day. NorthernMax is available in either a formaldehyde- or glyoxal-based gel format (NorthernMax and NorthernMax-Gly).
RNase-Free Reagents for Northerns
RNase contamination is a concern for any researcher working with RNA. Contamination of the reagents used to analyze RNA can result in degradation of the RNA and failed experiments. Several of the reagents and chemicals commonly used for the preparation of Northern assay solutions, as well as some commercially available premade solutions, have been shown to be frequently contaminated with RNase (Figure 2).
NorthernMax reagents are the same components included in the NorthernMax Kits, but in larger sizes. All NorthernMax reagents are supplied ready-to-use and are certified RNase-free. Comprehensive instructions for the use of these products are included.
ULTRAhyb™ Detects a Signal in Hours; Other Hybridization Solutions Require Days. Duplicate Northern blots were prepared using mouse thymus total RNA. Each blot was hybridized with DNA probe to p53 at a concentration of 1 x 106
cpm probe/ml hybridization solution. Incubation and washing conditions were as per protocol for each solution. Blots were exposed to film for the indicated times. Current Protocols in Molecular Biology, 1994. Ausubel, F.M. and others, Editors John Whiley & Sons, Inc. Molecular Cloning: A Laboratory Manual, 1989. Sambrook, J. Fritsch, E.F., Maniatis, T. Cold Spring Harbor Laboratory Press
Figure 2. RNase Testing of Northern Hybridization Solutions. 1 µl of each test solution was added to 9 µl of water containing 1 ng of a 5 x 104 cpm single-stranded RNA probe and incubated overnight at 37°C. 5 µl of each reaction were electrophoresed on a 5% acrylamide/8 M urea gel and exposed to film overnight. NOTE: Probe in water control lane shows radiolysis; use this control lane for comparisons to other lanes.
Optimized Hybridization for Oligonucleotide Probes
ULTRAhyb™-Oligo Hybridization Buffer is optimized specifically for hybridization of oligonucleotide probes to Northern blots. Figure 3 shows identical Northern blots that were hybridized overnight with a 32
P-labeled 44-mer oligonucleotide probe to ‹-actin in either ULTRAhyb-Oligo or a standard hybridization solution. The blots were exposed to film for 4 hr. Oligonucleotides ranging in length from 26 nt to 45 nt have been tested. ULTRAhyb-Oligo works best using oligonucleotides with predicted melting temperatures of 78-90°C and a GC content of 47-62%. The optimal hybridization temperature for oligonucleotide probes that differ in GC content and Tm values should be determined empirically.
Figure 3. ULTRAhyb™-Oligo vs. a Standard Hybridization Buffer Using DNA Probes. Duplicate Northern blots were prepared using the indicated amount of mouse spleen total RNA. Each blot was hybridized to the same 32P-labeled 44-mer oligonucleotide complementary to ‹-actin overnight using ULTRAhyb-Oligo or the standard hybridization solution described in Molecular Cloning (Maniatis). Blots were washed and exposed to film for 4 hours.