The MAXIscript® in vitro
Transcription Kit from Ambion was developed for the synthesis of highly specific radio- and non-isotopically labeled RNA probes used in hybridization reactions like ribonuclease protection assays, Northern and Southern blotting, and in situ
hybridization. In these in vitro
transcription reactions, RNA polymerases SP6, T7 and T3 are used. They are dependent on DNA as template and possess a high specificity for their respective 23 base promoters. The RNA polymerases of the MAXIscript® kits are very efficient in producing single-stranded full-length RNA probes with high specific activity, even when limited amounts of the radiolabeled nucleotide are present. According to Ambion, the MAXIscript® kits are also an excellent choice for the incorporation of modified nucleotides and the synthesis of nonisotopically labeled RNA probes in combination with Ambion's BrightStar® Psoralen-Biotin Labeling Kit.
Ambion provides different MAXIscript® in vitro transcription kits for DNA templates with T7, T3, or SP6 promoters. The kits contain quality tested DNase/RNase-free reagents: 10x Transcription buffer, 10 mM ATP/CTP/GTP/UTP solutions, TURBO DNAse™ I (2U/µl), dH2O and a gel loading buffer. A template DNA, pTRI-actin-mouse, for generation of a control mouse beta-acting probe is also included. The MAXIscript® Kit is available for 30 and 100 reactions, and is stable for 6 months, when stored at -20°C. When generating a RNA probe, it is important to synthesize the complementary anti-sense transcript of the mRNA to which it will hybridize.
I used the the MAXIscript® T7 Kit with different mouse cytokine and chemokine multiprobe templates from BD Biosciences (e.g. the multi-probe template mck-3b to detect mRNA of LTalpha, LTbeta, TNF, IL-6, IFNgamma, IFNbeta, TGFbeta1, TGFbeta2, TGFbeta3, MIF, L32 and GAPDH). These purified and ready-to-use multiprobe DNA templates can be used for T7 RNA polymerase-directed synthesis of [alpha32P]-labeled, antisense RNA probes, which hybridize with the mouse target mRNAs. If purifying your own plasmid DNA from bacteria, you will need to perform restriction digestion to linearize the DNA template and a proteinkinase K digestion to remove contaminating proteins.
In my initial trials, I used an increased volume of [alpha32P]-UTP with lower specific activity than recommended and I could not detect the probe after hybridization with mRNA from mouse brain. When I did not receive the expected signal for the hybridized RNA product, even after using a new probe and freshly isolated mRNA, I called Ambion’s technical service. They recommended to always use the higher specific activity, since a minimum of 3 µM of the radiolabeled nucleotide is needed to efficiently synthesize full-length RNA transcripts with less than 400 nucleotides. In addition, they sent me a slightly different protocol, which they optimized for the use with the multiprobe set templates from BD Biosciences, and indicated that it is important to add the reagents in the order specified in the protocol. In the protocol, 5 µl of [alpha32P]-UTP (3000Ci/mmol, 10 mCi/ml (3.3 µM)) was used in a 20 µl transcription reaction. For the transcription reaction, the samples were incubated for 1 hour at 37°C and than digested with 2 U DNase I for 30 min at 37°C to remove the DNA template. After addition of 80 µl dH2O, the DNA was extracted with 100 µl of phenol:chloroform:isoamyl alcohol (25:24:1). The DNA was precipitated from the supernatant of the extract with 1/10 volume of 5 M ammonium acetate and 2.5 volumes ethanol at -20°C and pelleted by centrifugation for 30 min at 4°C. The pellet was resuspended in 50 µl hybridization buffer. When following this new protocol, the effective incorporation of nucleotides during transcription could be monitored by measuring the counts of the incorporated radioactivity in 1 µl of the labeled probe in a scintillation counter. 2 µl of the probe containing 6 x 105 cpm were used for hybridization with 20 µg of total RNA isolated from mouse spinal cord and brain in the following RNase Protection Assay (RPA).
For a fast and efficient transcription reaction resulting in a highly-specific radiolabeled probe, I recommend the MAXIscript® in vitro transcription kit from Ambion. Except for the template DNA and radioactive nucleotide, the kit contains all reagents with an easy to follow protocol. Ambion’s website provides several technical bulletins, all protocols and a manual containing a troubleshooting section for using the kit. In addition, Ambion’s technical service has a lot of experience in working with RNA and was very helpful in optimizing my protocol.