The mRNA levels for each cDNA probe were normalized with respect to the plant glyceraldehyde-3-Pi dehydrogenase (GAPDH) mRNA levels, which has been validated as a reference gene for roots inoculated with P. indica ([ 92], and references therein).
The data obtained by PCR assay for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were validated as a reference gene by displaying the Ct values as box-and-whisker plots, and the distribution examined under the treatment conditions (data not shown).
The data obtained by PCR assay for GAPDH were validated as a reference gene by displaying the Ct values as box-and-whisker plots, and the distribution examined under mechanical loading conditions (data not shown).
The data obtained by PCR assay for Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) were validated as a reference gene by displaying the Ct values as Box and Whisker plots and the distribution examined under mechanical loading conditions (data not shown).
Finally, 4 further genes were added that were homologous to S. cerevisiae ALG9 (Sc1), TAF10 (Sc2), TFC1 (Sc3) and UBC6 (Sc4), which have been previously validated as good reference genes in this yeast [ 76].
The data obtained by PCR assay for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was validated as a reference gene by displaying the Ct values as box and whisker plots, and the distribution examined under free swelling and mechanical loading conditions (data not shown).
Our study demonstrates that the top six most stably expressed miRNAs (let-7a, miR-16, miR-26a, miR-345, miR-425 and miR-454) described herein should be validated as suitable reference genes in both high-throughput and lower throughput RT-qPCR colorectal miRNA studies.
Once the pons has been validated as a pseudo-reference region, reference-based modeling could be useful in providing a simple and robust alternative for BPND estimation without arterial sampling.
csrA (carbon storage regulator) and manB (phosphomannomutase) were used for normalization of bacterial qPCR data, as these had previously been validated as suitable bacterial reference genes for the tissue applied in the present study [ 23]. cDNA replicates were averaged after reference gene normalization.
In conclusion, the ubiquitin-conjugating enzyme 18 gene (UBC18) was validated as a suitable reference gene across all the plant samples examined.
