Hello!
We performed qRT-PCR on a specific gene and subsequently conducted bulk RNA-seq on the same samples.
In the qRT-PCR results, the gene showed over 2-fold higher expression in the case group compared to the control. However, in the RNA-seq analysis performed using Illumina, there was almost no difference, and the expression level of the case group was slightly higher than that of the control.
What could be the potential issues causing this discrepancy?
The read counts in the count matrix were similar across samples, and the overall read counts were consistent. Additionally, the data were normalized using DESeq2, and no outliers were observed in PCA or MDS plots.
The count matrix was generated through the following pipeline: FASTQ - > Trimming (Cutadapt) - > Alignment (HISAT2) - > Gene Mapping (featureCounts).
The quality at each step was good, so no additional QC was performed.
Thanks for your answer.
qPCR is often misinterpreted in my opinion. Statistics do not take into account the overall range of the Ct values. For example, fold changes are always higher and noisier, the more lowly a gene is expressed, meaning the higher the Ct values are. RNA-seq in contrast is aware of the non-equal relationship between variance and expression and takes this into account in the statistical analysis. Also, qPCR is often poorly-controlled, for example using a single "housekeeping" gene. In contrast, RNA-seq normalizes using thousands of genes, so I would generally trust RNA-seq more, unless qPCR is done well, with good experimental replication, validation primers of good effficiency and normalized against a good panel of reliable reference genes.
have you try convert counts to RPKM or FPKM or TPM