I have identified several technical and methodological concerns specific to the field of plant molecular biology and RT-qPCR reference gene validation.
Why were primer efficiencies not reported, and how were they validated?
How was biological replication handled—were samples pooled or analyzed individually?
Why were rRNA genes included despite known limitations in RT-qPCR normalization?
Was RNA quality assessed quantitatively (e.g., RIN values)?
How were primers validated for specificity in D. moldavica, given the lack of a published genome?
Why was reference gene validation not performed for all experimental conditions (e.g., different tissues, flower stages) using target genes?
What is the biological rationale for selecting HSP70 as a reference gene during flower development, given its typical role in stress response?