Quantitative Real-Time PCR Protocol

qRT-PCR was performed as previously reported [60 (link)]. Total cellular RNAs from iDC and 27DC were isolated as described above. 1 μg of RNA was used to make cDNA using the TaqMan Reverse Transcription Reagents (Thermo Fisher Scientific). Samples were run on Applied Biosystems GeneAmp PCR system 9700 at 25˚C for 10 minutes, 48˚C for 30 minutes, and finally for 95˚C for 5 minutes. DNA products were used for real time qRT-PCR reaction using the TaqMan 2X Universal PCR Master Mix (Thermo Fisher Scientific) on an iQ5 RT-PCR detection system (BioRad, Hercules, CA, USA) [61 (link)].

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Imamichi T., Chen Q., Sowrirajan B., Yang J., Laverdure S., Marquez M., Mele A.R., Watkins C., Adelsberger J.W., Higgins J, & Sui H. (2023). Interleukin-27-induced HIV-resistant dendritic cells suppress reveres transcription following virus entry in an SPTBN1, autophagy, and YB-1 independent manner. PLOS ONE, 18(11), e0287829.

Publication 2023

TaqMan Reverse Transcription Reagents GeneAmp PCR system 9700 TaqMan 2X Universal PCR Master Mix iQ5 RT-PCR detection system

Cdna Cellular Real time pcr Reverse transcription Rt pcr

Corresponding Organization : Frederick National Laboratory for Cancer Research

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Variable analysis

independent variables

RNA isolation method
Cell type (iDC and 27DC)

dependent variables

Gene expression levels

control variables

Amount of RNA used for cDNA synthesis (1 μg)
Reverse transcription conditions (25°C for 10 minutes, 48°C for 30 minutes, 95°C for 5 minutes)
Real-time qRT-PCR assay conditions (TaqMan 2X Universal PCR Master Mix, iQ5 RT-PCR detection system)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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