Question: How do I interpret the qPCR plot to determine the Sample Index PCR cycle number?
Answer: During the Visium Gene Expression for FFPE workflow, qPCR using the KAPA SYBR Fast qPCR Kit (KK4600) is performed to determine the appropriate cycle number for Sample Index PCR (SI-PCR).
Some qPCR systems require a negative reference dye (i.e., ROX) to be added when using the KAPA SYBR FAST qPCR Kit. Please reference the Roche KAPA SYBR ® FAST qPCR Kit Instrument Compatibility Table, or consult the real-time qPCR system manufacturer to determine whether your real-time qPCR instrument requires the use of ROX.
After the qPCR run has been completed, the threshold for determining the cycle number should be set at ~25% of the peak RFU value after baseline subtraction, or ~25% of the peak ΔRn if using ROX. Selecting the correct cycle number may require manually adjusting the RFU or ΔRn threshold depending on the software's settings for your qPCR system. We have provided step-by-step instructions below.
Step 1. Set the y-axis to a linear scale. Interpreting the plot using a log scale may lead to underestimating the cycle number. Plot RFU on the y-axis if not using a reference dye or ΔRn if using a reference dye. If using RFU, perform baseline subtraction if needed.
Step 2. Identify the peak RFU or ΔRn value.
Example: peak RFU = 8600 (dotted blue line)
Step 3. Calculate 25% of the peak RFU or ΔRn and use it as the threshold.
Example: threshold = peak value at 8600 RFU x 0.25 = 2150 RFU (green line)
Step 4. Find the cycle number at which the amplification curve intersects the threshold line.
For example, the threshold intersects the curve at ~14 cycles (dotted red line)
Step 5: If the cycle number is between two whole numbers, round accordingly.
Samples within +/- 2 cycle number can be combined in a single SI-PCR run, performing at the higher cycle number. We do not recommend combining samples if the cycle number difference is >2 to avoid library over-amplification.
Products: Visium for FFPE