Tips for cDNA Amplification and Library Construction of FFPE Samples with the VITA Platform
Since its debut in 2023, the VITA single-cell transcriptome platform has enhanced study of formalin-fixed paraffin-embedded (FFPE) samples, enabling researchers to extract high-resolution molecular insights from historically challenging archival specimens.
Through ongoing collaboration with users, we have refined VITA protocols to address critical bottlenecks in cDNA amplification and library preparation – stages where even minor technical variations can significantly impact sequencing outcomes. These steps demand particular attention when working with FFPE-derived material, where factors like nucleic acid fragmentation and inhibitor carryover pose unique challenges.
Effective cDNA amplification and library construction are critical in generating high-quality single-cell transcriptome data. This article shares valuable tips to streamline these steps on the VITA platform for FFPE samples, helping researchers obtain robust cDNA yields and reliable library quality.
Tip 1: Ensure Complete cDNA Recovery from Droplets
If the biphasic separation is incomplete, begin the cDNA process by inverting and briefly centrifuging on a mini centrifuge to ensure proper mixing.
Tip 2: Minimize Sample Loss During cDNA Recovery
During cDNA recovery, transfer the upper aqueous phase to a Filter Column (provided in the kit) and centrifuge for 30 sec to collect the filtrate. If liquid remains in the column, perform another brief spin to minimize sample loss.
Tip 3: Set Up a Negative Control for qPCR
Always prepare one extra qPCR mix beyond the actual sample count for the negative control. A negative control verifies that any detected signal comes from the sample, not background noise or contamination.
Tip 4: Select the Melt Curve Program in qPCR
Make sure to select the melt curve program when configuring the qPCR. An example melting curve is shown in the figure below. This allows us to evaluate the purity of the amplification product and detect any potential non-specific contamination, such as primer dimers.
Tip 5: Monitor qPCR Fluorescent Signal Peaks for Quality Control
Monitor the cycle number at which the qPCR fluorescent signal peaks to assess cDNA quality. Peaks appearing within 22 cycles indicate successful cDNA amplification (as shown in the figure below), while peaks at 23-24 cycles suggest potential risks. Peaks beyond 25 cycles are typically considered indicative of failure.
Tip 6: Optimize PCR Cycle Number for cDNA Amplification
Set the appropriate cycle for cDNA amplification based on the sample’s qPCR fluorescence peak. For optimal results, use between 20 and 23 cycles, adjusting according to the qPCR fluorescence data.
Tip 7: Check cDNA Fragment Size
After amplification, verify that the overall cDNA fragment sizes range from 100-1000 bp (example shown in the figure below). Fragments smaller than 100 bp should comprise less than 20% of the total. If small fragments dominate, perform a magnetic bead purification using a ratio of 0.8:1.
Tip 8: Verify Library Fragment Size for Sequencing
Ensure that library fragments range between 300-800 bp. Fragments smaller than 300 bp should comprise less than 20% of the total, with a single main peak showing a standard normal distribution (as shown in the figure below). If an excess of small fragments is present, perform a magnetic bead purification using a ratio of 0.7:1.
Tip 9: Limit Mixed Library Proportion in Next-Generation Sequencing
When sequencing, ensure that the selected library comprises no more than 30% of the total mixed library. This balance prevents bias in sequencing results and ensures high-quality data from all libraries.
These tips aim to help optimize cDNA amplification and library construction stages of VITA single-cell transcriptome experiments for FFPE samples. We hope this curated set of guidelines empowers researchers to maximize data quality and consistency, unlocking deeper insights in single-cell RNA sequencing of FFPE samples.
