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. 2018 Mar 23;13(3):e0194412. doi: 10.1371/journal.pone.0194412

Table 2. Comparison of RT-qPCR conditions using the pre-existing in-house RT-qPCR assay*.

Factor Mastermix# Sample volume Reaction volume Annealing temperature Primer concentration Probe concentration
I Mastermix 1. Superscript-III
2. Express
3. Fastvirus
5μl 1. 25μl
2. 20μl
3. 25μl
60°C 400nM 160nM
II Reaction volume Superscript-III 1. 5μl
2. 10μl
1. 25μl
2. 50μl
60°C 400nM 160nM
III Sample volume Superscript-III 1. 10μl
2. 16μl
50μl 60°C 400nM 160nM
Fastvirus 1. 5μl
2. 15μl
25μl 60°C 400nM 160nM

* All experiments were performed with G1-1326 RNA tenfold dilutions.

#Superscript-III kit: SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase; Express kit: EXPRESS One-Step SuperScript® qRT-PCR and Fastvirus kit: TaqMan® Fast Virus 1-Step.