coauthors reported that not all of the best known reference genes are equal. Further to this, Czechowski and coauthors showed that the most frequently used reference genes are hardly appropriate for data normalisation, and proposed a number of novel reference genes. To date, there are many studies in which the search and validation of reference genes are reported, but most of them are focusing on the traditionally used ��housekeeping��genes, not novel candidate reference genes that have been inferred from genomewide studies such as in. This issue can be settled by obtaining ortholog sequences for novel references with the help of degenerate primers, or by searching genome/transcriptome-wide sequencing data in addition to further validation. Moreover, even if reference genes have already been selected for the object, double-checking of their expression stability under experimental conditions is MedChemExpress Gynostemma Extract preferable in order to increase the accuracy of realtime qRT-PCR analysis. Another group of probable source of errors is more specific, but no less dangerous, and can result in incorrect data acquisition. The qRT-PCR data generation and analysis methodology indirectly implies that the samples being compared are similar in their morphology. The extent of the applicability of qRT-PCR to comparative analysis of gene expression levels in objects which are characterised by different morphology has never been discussed. We assume that in this case PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189346 the data obtained from real-time qRT-PCR Limited Applicability of qRT-PCR could be biologically meaningless. In order to test this, conclusions based on qRT-PCR data can be compared to those based on more direct experimental evidence such as in situ hybridisation or gene interactions predicted by mutant analysis. To investigate the influence of object morphology on the validity of qRT-PCR data we analysed the expression of genes involved in flower development and maintenance of floral meristem. Alteration of stem cell activity in the floral meristem in mutants of Arabidopsis thaliana is characterised by a dramatic change in floral organ number and identity. Results The expression levels of genes controlling floral organ identity, APETALA2, APETALA3 and PISTILLATA ) and regulators of meristematic activity and CLAVATA1, 2 ) were analysed in three mutants with an altered number and identity of floral organs: ap2-14, ag-1 and clv3-2. The qRT-PCR analysis of the ap2-14 mutant revealed that expression 
levels of AP3 and PI had decreased four-fold and threefold respectively. The expression level of AG had increased by slightly more than half. The expression levels of AP2, CLV1, CLV2 and WUS did not change significantly. 2 Limited Applicability of qRT-PCR 3 Limited Applicability of qRT-PCR Gene expression levels obtained from the analysis of ag-1 indicated that WUS expression was reduced by two orders of magnitude. While the expression levels of AP3 and PI had increased fourfold, and AP2 and CLV1 levels had tripled and doubled respectively.. In the clv3-2 mutant no significant changes in gene expression levels were observed except for PI, for which the expression level was reduced two-fold. Discussion Over the last twenty years, many aspects of the genetic control of development in Arabidopsis thaliana have been uncovered. In particular, the mechanism underlying the determination of floral organ identity and the system for the regulation of meristematic activity of the floral meristem. The functions and interactions of