Quantitative PCR

experimental objectives

To demonstrate the validity of Transcriptic’s Autoprotocol-inplemented qPCR Protocol.

methodology

Amplification primers were generated using the Oligosynthesis Quick Instruction.  Primers amplified a 265 bp amplicon encoded in the pUC19 plasmid (New England Biolabs).  PCR-derived amplicons were concentrated and purified into distilled water (ISOLATE II PCR and Gel Kit; Bioline) and DNA content determined (NanoDrop Lite Spectrophotmeter; Thermo Scientific).  DNA concentration was adjusted and serially diluted to generate four DNA standards(0.1; 0.01; 0.001; 0.0001 ng/µl).  The RTqPCR Protocol was performed using 2 µl of DNA standard (12x replicates per sample), 0.5 µlforward primer, 0.5 µlreverse primer (both 10 µM), 7.5 µl SensiFAST SYBR No-ROX Mix (Bioline) made up to 15 µlwith distilled water in each well. Thermocycling conditions were 98 °C, 3 minutes; [95 °C, 15 seconds; 60 °C, 15 seconds; 72 °C, 20 seconds]  x40.  Fluorescent measurements were recorded each cycle immediately after extension.

Results

Figure 1  (A) Amplification of DNA depicted as relative fluorescent units (RFU) vs. cycle number.  (B) Melt peak analysis ([derivative RFU / derivative temperature] vs. temperature).  Twelve replicates for each DNA standard were assessed.  (C) Statistical analysis of cycle threshold (Ct) data. S.D.: standrad deviation. S.E.M.: standard error of the mean. %C.V.: percentage coefficient of variance.  Error bars: S.E.M..