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Some reference genes such as ACTB and B2M are expressed somewhat more in stomach tissues than in cancer cell lines.
We have found that the coefficients for some reference genes are fairly large while they are close to zero for others (Additional file 2: Table S2).
This suggests that there may be artefacts of the standard-curve-based measurements for qPCR leading to higher copy numbers being observed for some reference genes compared with others.
By calculating ΔΔCq for each reference gene, and plotting these, in ascending order as normalised by a nominal reference gene, the user can see whether some reference genes show more similar expression to each other, and whether other genes stand out.
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It is noteworthy that, despite the fact that some reference-genes remain stable during microgravity exposure, several others, investigated in the study of C. S. Thiel et al., change quite dramatically, thus reinforcing the concept that exposure to near weightlessness may have a profound impact on living processes.
On the other hand, some single reference genes showed low expression stability.
XP-8 were first examined in this study although some traditional reference genes have been used for qRT PCR data normalization in other studies on Phomopsis liquidambari (Xie and Dai 2015).
Although some endogenous reference genes demonstrated high power of discrimination their ranking was dictated by previous knowledge of the expected experimental results (gp100 being melanocytic lineage specific).
Estimates of the copy number of the SR3 retrotransposon were established by a comparative bioinformatics approach [ 12- 14] wherein BLAST analysis of the BAC-end database of S. mansoni genomic sequences targeted more well-characterized retrotransposable elements from S. mansoni, and some other reference genes, for which copy numbers have been reported.
We had anticipated that more neuronal specific genes would exhibit a decreasing pattern, but noted that even some common neuronal reference genes, such as ENO2 (ranking 7597th), were not associated with age.
Analysis of the raw expression levels across all samples detected some variation among reference genes.
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