Micro RNAs as reference genes for quantitative gene expression studies using RT-qPCR in cotton

Functional Genomics

poster

Fausto, Anna Karoline Silva; Silva, Tatiane ; Romanel, Elisson; Vaslin, Maite ; Fausto, Anna Karoline Silva; Silva, Tatiane ; Romanel, Elisson; Vaslin, Maite

Vaslin, Maite ; Vaslin, Maite

Vaslin, Maite ; Vaslin, Maite

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a technique largely used in the investigation of gene expression. However, for correct analysis and interpretation of their results, the choice of a suitable gene to normalize the data is a crucial factor. The genes used to normalize expression data, called as reference genes, most have constant expression levels in different tissues and across different conditions. However find genes stable in all situations and that work at the same form in different plant species and conditions is not an accessible goal. The technique of second generation sequencing has contributed to the identification a number of new genes, including microRNAs (miRNAs). miRNAs have important regulatory roles in eukaryotic acting in at post-trasncription level at different biological functions. In the present study, we evaluated, for the first time cotton, micro RNAs as candidates as reference genes for cotton gene expression studies. The stability of miRNAs in Gossypium hirsutum in different tissues (root, stem, leaf and flower), different cultivars (FiberMax FM966, Delta Opal and Cedro) and under biotic stress caused by infection of Cotton leaf roll dwarf virus (CLRDV) was analyzed. mRNAs already described as reference genes in cotton were also included in these analyzes. Cotton samples were organized in 12 sets where the expression stabilities of the six miRNAs and five mRNA reference genes were evaluated. Four algorithms, geNorm, NormFinder, BestKeeper and ΔCt were used to identify the stability of these genes and therefore provide an accurate selection of reference genes. In 8 of the 12 sets tested, the micro RNA (miRNAs) were the best housekeeping or reference genes. After found the best reference genes between the micro RNAs and the mRNAs for these twelve experimental conditions, we validated them in expression assays performed in study cases. For that, we studied the expression of miRNAs and mRNA already know to show distinct expression levels in the analyzed experimental conditions. Study cases used leaves of “cv FM966” under stress biotic caused by Cotton blue disease and flower x leaves from FM966 and Cedro. miR2910, miR164, miR2118, miR2111, miR3476, miR159, GhDCL1, GhDCL2, GhDCL3 and GhDCL4 were evaluated under biotic stress and miR164, GhPGFS7 and GhXTH expressions levels were evaluated in leaves and flowers. Ours results show that the reference microRNA/mRNA genes previously selected work as excellent reference genes in all of the study cases. Statistical teste p sustained the expression levels of the differentially expressed miRNA and mRNAeven under biotic stress as in flower x leaves. These analyzis also showed that to normalize target mRNA the origin, miRNA or mRNA, of the reference gene is not important, but to normalize miRNA expression levels, miRNA reference genes seems to be better than mRNAs. The indication that for miRNA expressions analyzes we should use miRNA as reference genes instead of mRNAs was already well supported in human miRNAs studies, however this is the first study were this is shown in plants. Financial Support: CAPES and FAPERJ