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    RNA extraction, cDNA synthesis and Taqman qPCR

    gurvir.virdi

    Published: 2022-09-20 DOI: 10.17504/protocols.io.36wgqj5rkvk5/v1
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    Abstract

    RNA extraction, cDNA synthesis and TaqMan qPCR on samples.

    Steps

    RNA extraction

    1.

    Cell pellets are snap-frozen using dry ice.

    2.

    RNA is extracted using the Maxwell® RSC simply RNACells kit (Promega), and the Maxwell® RSC 48 instrument, following manufacturer instructions.

    3.

    After RNA extraction, RNA concentration and quality are measured and assessed using a nanodrop.

    cDNA synthesis

    4.

    Up to 1 µg of RNA per sample is reverse-transcribed into cDNA using the High-Capacity cDNA Reverse Transcription kit (ThermoFisherScientific).

    quantitative PCR

    5.

    qPCR is performed using TaqManTMGene Expression Assay (Thermo Fisher Scientific) according to the manufacturer’s instructions.

    6.

    For each gene, TaqManTMprobes were used along with the TaqManTMmaster mix, and sample cDNA following the manufacturer's protocol.

    7.

    Samples, along with a minus reverse transcriptase control (-RT) were ran for each gene on the QuantSudio 6 Flex Real-Time PCR System (Applied Biosystems).

    8.

    The -RT served as a negative control, and the gene expression levels were normalised to the housekeeping gene GAPDH following the delta-delta Ct method. Gene expression values were expressed as the normalisation to their respective control sample.

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