I recently conducted cDNA synthesis followed by conventional PCR for quality assessment. In the course of this experiment, I observed some unexpected results that have raised questions about the reliability of my cDNA and its potential impact on real-time PCR experiments.
Specifically, here are the key observations:
My primary concern is whether these observations in the cDNA synthesis and conventional PCR could potentially impact the outcomes of my real-time PCR experiments. Real-time PCR requires high precision, and any issues with cDNA quality or PCR variability could affect the accuracy of gene expression quantification.
I would greatly appreciate insights from the research community regarding the possible reasons for these observations and their potential implications for downstream real-time PCR experiments. Your expertise and suggestions on troubleshooting or optimizing this process would be invaluable.
If you want reliable results:
1. Always use internal positive control, ideally before the RT step (same with NTC)
2. Do everything in triplicates whenever possible
3. Run standard curve during qPCR
With that in mind, sample differences during qPCR can be the result of mismatching primers/SNPs in the primer region
Not really sure what your problem is due to lack of details, but yes cDNA synthesis (via reverse transcription) is a known step that results in biases/ artifacts and confound the resulting cDNA mixture.
https://pubmed.ncbi.nlm.nih.gov/36990512/
https://www.nature.com/articles/s41598-020-65005-0
Since the RT- control had amplification, I would think that you have a contamination somewhere. Start with fresh buffers and components. also the 260/230 ratio might give you more insight into your RNA quality as it indicates organic contamination and lower ratios for the samples that did not amplify well would support that. Also you don't mention if it is random primed cDNA or oligo(dT) as that would make a difference also depending on the location of the PCR primers and length of your target gene
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