Quantitative Real-Time PCR Protocol

Quantitative Real-Time Polymerase Chain Reaction (qPCR) was performed using the Accuris qMAX One-Step RT-qPCR kits (Accuris Instruments, Edison, NJ, USA) on the Applied Biosystems’ Quant Studio 5 platform (PE Applied Biosystems, Waltham, MA, USA). The experiments used a 25 µL reaction volume and thermo profile as shown in Table 2. The relative gene expression was calculated using the delta-delta thresh-hold cycle (ΔΔCt) formula ΔCt = Ct (gene of interest)—Ct (housekeeping gene) according to [20 (link)]. All primer sets used in the study are listed in Table 3.

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Mudenda M., Kimani J., Kinyua J, & Kimotho J. (2022). Preliminary In Vivo Evidence of Oral Selenium Supplementation as a Potentiating Agent on a Vector-Based COVID-19 Vaccine in BALB/c Mice. Vaccines, 11(1), 57.

Publication 2022

Quant Studio 5 platform

Gene Gene expression Housekeeping gene Primer Quantitative real time polymerase chain reaction

Corresponding Organization : Kenya Medical Research Institute

Other organizations : Jomo Kenyatta University of Agriculture and Technology

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Relative gene expression calculated using the delta-delta thresh-hold cycle (ΔΔCt) formula

control variables

Reaction volume (25 µL)
Thermal profile as shown in Table 2
Housekeeping gene used as a reference for ΔΔCt calculation

controls

No positive or negative controls were explicitly mentioned in the input protocol

Annotations

Based on most similar protocols

Use of a one-step RT-qPCR kit for reverse transcription and qPCR in a single reaction to minimize handling steps (Input Protocol)

Reaction volume used was 25 µL (Input Protocol)

Thermal profile for qPCR reaction is provided in Table 2 (Input Protocol)

Relative gene expression calculated using the ΔΔCt method (Input Protocol, Protocol 1, Protocol 2, Protocol 5)

Use of five housekeeping genes (ACTB, B2M, GAPDH, HPRT1, RPLP0) for normalization of gene expression data (Protocol 1)

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