Optimizing cDNA Amplification for Prokaryotic Samples: Practical Tips for the VITA Platform
With the VITA Single-Cell Transcriptome Platform, M20 Genomics has introduced a variety of high-throughput single-bacterial transcriptome kits offering new avenues and deeper insights in prokaryotic research. In this article, we will focus on practical tips for cDNA amplification to streamline workflows and improve experimental outcomes. We hope these guidelines help users achieve smoother and more efficient use of the VITA platform.
Tip 1: Achieve complete cDNA recovery
Incomplete mixture or not enough PFO during cDNA recovery can result in reduced cell yield. To address this, first invert the tube gently but thoroughly to mix the contents. Then, use a mini centrifuge to briefly spin down the mixture. This step helps separate the aqueous phase more effectively. Ensure that the aqueous phase is clearly separated before proceeding to avoid cross-contamination with oil residues.
Tip 2: Minimize sample loss during cDNA recovery
For maximum recovery of the aqueous phase after demulsification, carefully transfer the upper aqueous layer to a spin column without disturbing the oil layer and centrifuge for 30 seconds using a mini centrifuge to collect the filtrate. If residual liquid remains in the column, repeat centrifugation briefly to ensure complete recovery. Be sure to avoid excessive handling or delays during this step, as prolonged exposure can lead to evaporation or degradation of sensitive components.
Tip 3: Include a negative control during qPCR
Negative controls are essential to detect contamination or nonspecific amplification. Ensure the validity of qPCR results by preparing a reaction mix with DNase/RNase-free water as template for the negative control. Monitor amplification curves during qPCR to ensure no signal is detected in the negative control well. If amplification occurs in the negative control, review reagent preparation and handling procedures to identify potential contamination sources.
Tip 4: Include melting curve in qPCR program setup
A melting curve analysis serves to assess product specificity. Ensure that the qPCR program includes a melting curve step after amplification cycles. An ideal melting curve (example in figure below) shows a single peak is present, indicating specific amplification.
Tip 5: Ensure efficient qPCR cycle thresholds
The cycle threshold (Ct) value is an important indicator of amplification efficiency. Peaks observed within 22 cycles are considered optimal (example in figure below). Peaks at 23–24 cycles suggest potential issues with template quality or reaction efficiency and require careful review. Peaks at ≥25 cycles indicate suboptimal conditions, such as low template concentration or poor reaction setup, and are unacceptable for subsequent steps.
Tip 6: Use appropriate cycle number for cDNA amplification
Proper cycle settings during cDNA amplification ensure the generation of high-quality libraries. To achieve this, set the program cycle number between 15–23, depending on input material and template quality. Select the cycle number corresponding to the highest fluorescence value in qPCR fluorescence curve as a reference point. Be sure to avoid excessive cycling, as this can lead to over-amplification and introduce artifacts into your library.
Tip 7: Assess cDNA fragment size distribution
Evaluating cDNA fragment size ensures library quality and sequencing compatibility. The overall fragment size of amplified cDNA should range between 100 bp and 1000 bp in size. Additionally, fragments between 100–200 bp should account for less than 20% of total fragments (example in figure below). If smaller fragments dominate, remove them using magnetic beads at a bead-to-cDNA volume ratio of 0.8:1.
Tip 8: Verify library fragment size after construction
Proper library fragment size distribution is essential for sequencing success. Constructed libraries should utilize fragments ranging between 300–800 bp in size. Fragments smaller than 300 bp should comprise less than 20% of total fragments. The library should exhibit a single main peak with a standard normal distribution (example in figure below). If smaller fragments dominate, remove them using magnetic beads at a bead-to-cDNA volume ratio of 0.7:1.
Tip 9: Adjust library mixing ratios for sequencing
To prepare libraries for high-throughput sequencing, select the library or libraries that have successfully passed the quality control for next-generation sequencing. Ensure that the library constitutes no more than 15% of the total mixed library used for sequencing. This prevents over-representation of any single library and ensures accurate sequencing across samples.
With these tips, we aim to provide practical guidance for optimizing cDNA amplification workflows in prokaryotic samples using M20 Genomics' VITA platform. By following these recommendations, researchers can enhance experimental efficiency and data quality. We hope these insights serve as valuable tools in your scientific endeavors, paving the way for fruitful discoveries in prokaryotic research.
Stay tuned for future updates, where we'll share additional tips to help you further enhance experimental efficiency and improve data quality when using VITA single-cell transcriptome products.
