This step utilizes Anchor PCR primers to amplify the target cDNA fragments flanked with the Anchor 1 and Anchor 2 sequences generated during the previous Reverse Gene-Specific Primer Extension step.

  1. Prepare the Anchor PCR Master Mix as shown below for all samples and controls plus 5% extra volume of all components as a safeguard against pipetting error:

    Anchor PCR Master Mix Component Volume per sample, µl
    Anchor Primer Mix 10
    PCR Buffer 13
    dNTP Mix 1.3
    PCR-Grade Water 40
    DNA Polymerase 0.7
    Total 65
  1. Gently vortex Master Mix, and spin down briefly to collect droplets. Spin down the Reverse GS Primer Extension plate, remove the seal, then add 65 μl of Anchor PCR Master Mix to each reaction well:

    Component Volume per sample, µl
    Reverse Gene-Specific Primer Extension DNA (previous step) 35
    Anchor PCR Master Mix (prepared above) 65
    Total 100
  1. Seal the plate with new adhesive film and spin down to collect droplets.
  1. Load the plate in the thermal cycler. Run the following program, using the recommended number* of PCR cycles:

    Temperature Time Cycles
    98°C 30 sec 1
    98°C 10 sec 14-26
    * see table below
    72°C 20 sec
    72°C 30 sec 1
    4°C 1
  • To avoid bias in gene expression levels due to overcycling, refer to the table below which provides the recommended number of PCR cycles based on the starting amount of RNA used for cDNA Synthesis. The recommended number of cycles may vary depending on the specific DriverMap panel used.

    Starting
    RNA
    Amount
    (ng)
    (1) hGW V2
    (2) hHallmark
    (3) Mouse Targeted
    (1) hPan-Cancer
    (2) hTransFactor
    (3) hLINCSx
    (1) hCell Marker
    Total RNA
    RIN > 5
    (from cells or
    fresh tissue)
    0.01 26 27 28
    0.05 24 25 26
    0.2 22 23 24
    0.8 20 21 22
    3 18 19 20
    12 16 17 18
    50 14 15 16
    FFPE
    RNA
    50 20 21 22
    300 16 17 18

NOTE: For mRNA samples prepared using the DirectCell™ Oligo-dT Capture Protocol, we recommend running at least 16 cycles when starting with thousands of cells per well, up to ~23 cycles for single cells.


Last modified: Feb 28, 2019

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