The Assay

The principle of fluorescence-based real-time RT-PCR assays is simple: reverse transcription of RNA is reverse-transcribed into cDNA; a suitable detection chemistry reports the presence of PCR products; an instrument monitors the amplification in real time; and an appropriate software analyzes the data.[7] Because the quality of the RNA template is the single most important determinant of the reproducibility of RT-PCR results,[8] it is essential to ensure that no inhibitors copurify during the RNA extraction process.[9]

Real-time RT-PCR can be either a one-tube assay using a single buffer, or a two-tube assay where both first-strand cDNA synthesis and the subsequent PCR step are performed separately under optimal conditions for the respective polymerases. The former is more convenient and reduces the risk of cross-contamination;[10,11] the latter may be more sensitive and more reproducible.[12]

The priming of the cDNA reaction from the RNA template is best performed using oligo-dT or target-specific primers. Although random primers yield the most cDNA, they initiate transcripts from multiple points along the RNA, including ribosomal RNA (rRNA), thus producing more than one cDNA per original target. Oligo-dT priming results in a faithful cDNA representation of the mRNA

pool, but it is not a good choice for poor-quality RNA from formalin-fixed archival material. Target-specific primers synthesize the most specific cDNA and provide the most sensitive method of quantification,[13] but require separate priming reactions for each target.

Viral RTs, used mainly in two-step assays, have a relatively high error rate and a strong tendency to pause, hence producing truncated cDNA.[14] Avian Myoblastosis Virus-RT (AMV-RT) is more robust and processive than Moloney Murine Leukemia Virus-RT (MMLV-RT)[15] and retains significant polymerization activity up to 55°C,[16] whereas native MMLV-RT has significantly less RNaseH activity than native AMV-RT[17] but is less thermostable. Several DNA-dependent DNA polymerases exhibit both RNA- and DNA-dependent polymerization activities in the presence of Mn2+.[18,19] It is also possible to use blends of reverse transcriptases in RT-PCR reactions, which can result in higher reverse transcription efficiencies than the individual component enzymes.

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Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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