4.4. PCR with Anchor Primers

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

2. 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

3. Seal the plate with new adhesive film and spin down to collect droplets.

4. 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.

*Stopping Point in procedure: PCR products may be stored at 4°C overnight.