Article Title: Template-dependent multiple displacement amplification for profiling human circulating RNA
Figure Lengend Snippet: Real-time reverse transcription–template dependent multiple displacement amplification (RT-tdMDA) using three hepatitis C virus (HCV) patient serum samples and a negative control (H 2 O) These samples covered the range of the RNA yield extracted from 200 μL serum (7.5–19.2 ng), as quantitated in the final 14 μL of elution by the QIAGEN miRNA kit prior to HL-DNase digestion. An aliquot of 10.6 μL RNA was used for RT in a reaction containing 200 U SuperScript III, 80 μM 5′-end-blocked random pentamer primer (5′-/iSpC3/NNN*N*N-3′; asterisks denote phosphorothioate bonds), and 2 mM dNTPs in a 20-μL volume. An aliquot of 4 μL of the RT reaction was used in a 40-μL tdMDA reaction containing 300 U phi29 DNA polymerase (Epicentre), 80 μM primer, and 0.1× SYBR Green I (Thermo Fisher Scientific). The reaction was incubated at 28°C for 24 h on the ABI TaqMan 7500, in which fluorescent intensities were monitored through the SYBR Green channel. Estimated amount of cDNA input in tdMDA = [10.6 / (14 + 0.5 (HL-DNase) + 1.4 (buffer)] × [total RNA amount] × 0.2. Template-independent amplification was completely inhibited, as indicated by the negative control.
Article Snippet: First, using the standard MDA protocol, we evaluated the purity of phi29 DNA polymerases from various vendors, including Lucigen (Middleton, WI), Epicentre (Madison, WI), Thermo Scientific (St. Louis, MO), and New England Biolabs (Ipswich, MA).
Techniques: Multiple Displacement Amplification, Negative Control, SYBR Green Assay, Incubation, Amplification