Integrated system simplifies and improves Northern blot analysis
Karen Dolter Jeff Braman
Streamline RNA analysis with a whole host of products from Stratagene. First, the StrataPrep total RNA miniprep kit is used to isolate ample yields of high-quality RNA from tissues and cells, then the RNA is ready to use in a number of applications, including Northern blot analysis. This procedure features the following products: the Joule Box minigel electrophoresis system, the Stratalinker UV crosslinker,* the prostar first strand RT-PCR kit, Stratagenes primer sets for RT-PCR, the StrataPrep PCR purification kit or the StrataPrep DNA gel extraction kit, the Prime-It II random primer labeling kit, NucTrap probe purification columns,** QuikHyb hybridization solution, and the PersonalHyb hybridization oven. When used together for Northern blot analysis, this excellent integrated system permits high sensitivity and reproducible results.
Northern blot analysis of gene transcription is dependent on such factors as isolating intact, high-quality RNA, probe-labeling to high specific activity, removing unincorporated radiolabeled nucleotides, and hybridizing with conditions that maximize the signal-to-noise ratio. Products developed at Stratagene eliminate many variables that lead to poor quality Northern blot results. Consequently, we used these products for a Northern blot analysis of MSH2 gene transcription in a hamster cell model system.
MSH2 is important for DNA mismatch repair, and mutations in this gene are responsible for many cases of hereditary nonpolyposis colorectal cancer.1 The MSH2 gene encodes a 3.1-kb mRNA that is expressed at low to moderate levels.2 The larger-than-average size and modest expression level of this RNA make it a good candidate for evaluating products for Northern blot analysis.
Stratagenes products for optimized Northern blot analysis (Figure 1) demonstrate substantial benefits: The StrataPrep total RNA miniprep kit allows pure total RNA to be isolated quickly and easily with high yields. Gels prepared with the Joule Box minigel electrophoresis system run fast and generate consistent results. Capillary transfer of RNA is efficient with the Duralon-UV membrane, and the Stratalinker enables rapid and simple immobilization of RNA on the membrane.
The cDNA synthesized with the ProSTAR first strand RT-PCR kit is suitable for many uses (including PCR). Stratagenes PCR primers, including primers for MSH2 transcripts, are optimized for RT-PCR. An MSH2 fragment is readily generated from cDNA during PCR using these primers. With the StrataPrep PCR purification or DNA gel extraction kit, DNA fragments purified after PCR are ready to be used in primer labeling reactions.
The Prime-It II random primer labeling kit is used to label DNA fragments to high specific activity in just a few minutes. Unincorporated nucleotides can be readily and swiftly removed from these probes using NucTrap probe purification columns. When hybridization of these probes is performed in QuikHyb solution, the hybridization occurs rapidly and with minimal background. The PersonalHyb hybridization oven is used with various sizes of tubes, avoiding the tedious manipulations required when using sealed bags for hybridization reactions. Additionally, the accurate and precise temperature control of this oven assures consistent results.
Use Stratagenes products for Northern blot analysis to achieve outstanding results with substantial time sav ings. In particular, UV-crosslink, label a probe, prehybridize, and hybridize the probe to the blot in less than 1.5 hours. For even greater convenience and consistent results in hybridization, use the QuikHyb solution in conjunction with the PersonalHyb oven. Altogether, Stratagene makes available an integrated group of products that simplifies Northern blot analysis and leads to quick and reliable results.
Isolation, Electrophoresis, and Blotting of Total RNA: Total RNA was isolated from 5 x 106 CHO cells using the StrataPrep total RNA miniprep kit.3 Duplicate samples containing 2, 1, 0.5, 0.2, and 0.1 mg of this RNA were electrophoresed on a 1.2% formaldehyde-agarose gel using Stratagenes Joule Box minigel electrophoresis system. The gel was blotted to Stratagenes Duralon-UV membrane4 in 10X SSC overnight. The membrane was air-dried and UV-crosslinked in the Stratalinker unit.4
Probe Labeling: Total RNA from CHO cells was used with the ProSTAR first strand RT-PCR kit5 to synthesize cDNA. An MSH2 fragment was generated from this cDNA by PCR using Taq2000 DNA polymerase and Stratagenes MSH2 primer set for RT-PCR6 in duplicate reactions, according to instructions. The expected single 0.9-kb band was generated. One of the reactions was purified using the StrataPrep PCR purification kit, and the other was purified with the StrataPrep DNA gel extraction kit.7 Each of these fragments was labeled with a-32P-dCTP in a 5-minute reaction using Stratagenes Prime-It II random primer labeling kit.8 The probes were purified to remove unincorporated nucleotides using Stratagenes NucTrap probe purification columns.9 The specific activity of each probe was greater than 109 cpm/g.
Hybridization of the Probes to Northern Blot s: The Northern blot containing total RNA from CHO cells was cut into duplicate sections, and each was prehybridized in 3 ml of Stratagenes QuikHyb hybridization solution for 15 minutes in Stratagenes PersonalHyb hybridization oven, according to the QuikHyb manuals instructions. Each blot was hybridized with 3 x 106 cpm of one of the probes (106 cpm/ml) for 1 hour, also according to the manual. The membranes were washed twice for 10 minutes in 2X SSC/0.1% SDS at room temperature and twice for 15 minutes with 0.1X SSC/0.1% SDS at 60C. The membranes were enveloped in plastic wrap and exposed to Kodak XAR-5 film at -80C (with an intensifying screen) for varying amounts of time. An overnight exposure detected the low to moderate-abundance MSH2 transcript from as little as 200 to 500 ng of total RNA. After a 4-day exposure, the expected 3.1-kb band was observed from as little as 100 ng of total RNA, with a minimum of background (Figure 2).
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* U.S. Patent Nos. 5,288,647 and 5,395,591 and 5,814,523
** U.S. Patent Nos. 5,336,412 and 5,378,360