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[DESCRIPTION]
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<p>The SensiFAST™ Probe Hi-ROX One-Step Kit has been formulated for highly reproducible first-strand cDNA synthesis and subsequent real-time PCR in a single tube. The kit is formulated for use with probe-detection technology, including TaqMan®, Scorpions® and molecular beacon probes. A combination of the latest advances in buffer chemistry together with a reverse transcriptase and hot-start DNA polymerase system, ensures that SensiFAST Probe Hi-ROX One-Step Kit produces fast, highly-specific and ultra-sensitive one-step real-time RT-PCR.</p>
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<p>The SensiFAST Probe Hi-ROX One-Step Kit consists of a 2x SensiFAST Probe Hi-ROX One-Step mix, separate reverse transcriptase and RiboSafe RNase Inhibitor.</p>
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[GUIDELINES]
<p><strong>Kit Components</strong></p>
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<p><strong>Reagent</strong></p>
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<p><strong>100 x 20 μL reactions</strong></p>
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<p><strong>500 x 20 μL reactions</strong></p>
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<p>SensiFAST™ Probe Hi-ROX One-Step mix (2x)</p>
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<p>1 x 1 mL</p>
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<p>5 x 1 mL</p>
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<p>RiboSafe RNase Inhibitor</p>
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<p>1 x 40 μL</p>
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<p>1 x 200 μL</p>
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<p>Reverse transcriptase</p>
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<p>1 x 20 μL</p>
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<p>1 x 100 μL</p>
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<p>DEPC-H<sub>2</sub>O</p>
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<p>1 x 1.8 mL</p>
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<p>2 x 1.8 mL</p>
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<p><strong>Instrument compatibility</strong></p>
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<p>The SensiFAST Probe Hi-ROX One-Step Kit has been optimized for use with all probe chemistries, including TaqMan, FRET, Scorpions and molecular beacon probes on real-time PCR instruments listed in the following compatibility table, each of these instruments having the capacity to analyze the real-time PCR data with the passive reference signal either on or off. The kit is also compatible with several instruments that do not require the use of ROX, such as the Qiagen (Corbett) Rotor-Gene™ 6000, the Bio-Rad CFX96 or the Roche LightCycler® 480.</p>
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<p><strong>Manufacturer</strong></p>
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<p><strong>Model</strong></p>
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<p>ABI (Invitrogen)</p>
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<p>7000, 7300, 7700, 7900, 7900HT, StepOneTM and StepOne™ Plus</p>
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<p><strong>General considerations</strong></p>
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<p>When handling RNA, it is important to use RNase-free plasticware and reagents. We also recommend performing RNA work in an RNase-free area. To help prevent any carry-over DNA contamination, we recommend that separate areas are maintained for reaction set-up, PCR amplification and any post-PCR gel analysis. It is essential that any tubes containing amplified DNA product are not opened in the reaction set-up area.</p>
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<p><strong>Primers and probe:</strong> These guidelines refer to the use of dual-labeled probes. Please refer to the relevant literature when using other probe types. The sequence and concentration of the probe and primers, as well as amplicon length, can be critical for specific amplification, yield and overall efficiency of any real-time RT-PCR.</p>
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<p>We strongly recommend taking the following points into consideration when designing and running your real-time RT-PCR:</p>
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<li>use primer-design software, such as Primer3 or visual OMP™ (<a href="http://frodo.wi.mit.edu/primer3/ " target="_blank">http://frodo.wi.mit.edu/primer3/ </a>and DNA Software, Inc. <a href="http:// dnasoftware.com/" target="_blank">http:// dnasoftware.com/</a>, respectively). Primers should have a melting temperature (Tm) of approximately 60°C. The Tm of the probe should be approximately 10°C higher than that of the primers</li>
<li>optimal amplicon length should be 80-200 bp, and should not exceed 400 bp</li>
<li>final primer concentration of 400 nM is suitable for most probe reactions. However, to determine the optimal concentration we recommend titrating in the range 0.2-1 μM</li>
<li>use an equimolar primer concentration</li>
<li>a final probe concentration of 100 nM is suitable for most applications. We recommend that the final probe concentration is at least 2-fold lower than the primer concentration</li>
<li><em>Note: In multiplex real-time RT-PCR, probe concentrations in excess of 100 nM can result in cross-channel fluorescence</em></li>
<li>where possible, use intron-spanning primers to avoid amplification from genomic DNA</li>
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<p><strong>Template:</strong> It is important that the RNA template is intact and devoid of DNA or contaminating inhibitors of both reverse transcription and PCR. For high purity RNA, we recommend using the Bioline ISOLATE II RNA Mini Kit (BIO-52073). RNA stocks and dilutions should be made in DEPC-treated Water to avoid any RNase-mediated degradation.</p>
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<p>The recommended amount of template for one-step real-time RT-PCR is dependent upon the type of RNA used:</p>
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<li>total RNA: purified total RNA can be used in the range from 1 pg to 1 μg per 20 μL reaction</li>
<li>mRNA: purified mRNA can be used from 0.01 pg per 20 μL reaction</li>
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<p><strong>MgCl<sub>2</sub>:</strong> The MgCl<sub>2</sub> concentration in the 1x reaction mix is 3 mM. In the majority of real-time RT-PCR conditions this is optimal for both the reverse transcriptase and the hot-start DNA polymerase. If necessary, we suggest titrating the MgCl<sub>2</sub> to a maximum of 5 mM.</p>
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<p><strong>RT-PCR controls:</strong> It is important to detect the presence of contaminating DNA that may affect the reliability of the data. Always include a no-RT control reaction, by omitting the reverse transcriptase from the reaction.</p>
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<p><strong>Optional ROX:</strong> The SensiFAST Probe Hi-ROX One-Step Kit is premixed with ROX (5-carboxy-X-rhodamine, single isomer), so that ROX fluorescence can be optionally detected on certain real-time instruments. If your real-time PCR instrument has the capability of using ROX and you wish to use this option, then this option must be selected by the user in the software.</p>
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<p><strong>Troubleshooting Guide:</strong><br />See the Bioline full documentation for detailed troubleshooting instructions.</p>
<p><a href="http://www.bioline.com/us/downloads/dl/file/id/3301/sensifast_probe_hi_rox_one_step_kit_manual.pdf" target="_blank">http://www.bioline.com/us/downloads/dl/file/id/3301/sensifast_probe_hi_rox_one_step_kit_manual.pdf</a></p>
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[STEPS]
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