Selection of Reference Genes for Normalization of MicroRNA Expression by RT-qPCR in Sugarcane Buds under Cold Stress

Abstract

Sugarcane, accounting for 80% of world's sugar, originates in the tropics but is cultivated mainly in the subtropics. Therefore, chilling injury frequently occurs and results in serious losses. Recent studies in various plant species have established microRNAs as key elements in the post-transcriptional regulation of response to biotic and abiotic stresses including cold stress. Though, its accuracy is largely influenced by the use of reference gene for normalization, quantitative PCR is undoubtedly a popular method used for identification of microRNAs. For identifying the most suitable reference genes for normalizing miRNAs expression in sugarcane under cold stress, 13 candidates among 17 were investigated using four algorithms: geNorm, NormFinder, deltaCt, and Bestkeeper, and four candidates were excluded because of unsatisfactory efficiency and specificity. Verification was carried out using cold-related genes miR319 and miR393 in cold-tolerant and sensitive cultivars. The results suggested that miR171/18S rRNA and miR171/miR5059 were the best reference gene sets for normalization for miRNA RT-qPCR, followed by the single miR171 and 18S rRNA. These results can aid research on miRNA responses during sugarcane stress, and the development of sugarcane tolerant to cold stress. This study is the first report concerning the reference gene selection of miRNA RT-qPCR in sugarcane.

RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya

“…Primer amplification efficiency indicates the amplicon doubling rate of a specific primer pair during a PCR, which affects the accuracy of RT-qPCR data [4,40]. A good primer combination has an E value close to 100% [34,35]. In our study, the E values of the primers used to amplify the five candidate reference genes were between 94% and 98.5%.…”

“…In these studies, it is vital to choose appropriate reference genes for obtaining accurate and reliable results. A good primer combination has an E value close to 100% [34,35]. However, there has been no systematic and accurate validation of these genes under various experimental conditions.…”

“…Primer specificity was checked by melting curve and agarose gel electrophoresis analyses. Primers were only used in subsequent experiments if the condition 90% ≤ E ≤ 110% was met [34,35], and the mean squared error of the single data points fit to the standard curve (Error) value was below 0.2, as recommended by the Light-CyclerÒ 480 Software user manual. Primers were only used in subsequent experiments if the condition 90% ≤ E ≤ 110% was met [34,35], and the mean squared error of the single data points fit to the standard curve (Error) value was below 0.2, as recommended by the Light-CyclerÒ 480 Software user manual.…”

Identification of Appropriate Reference Genes for Normalizing miRNA Expression in Citrus Infected by Xanthomonas citri subsp. citri

“…The Illumina HiSeq2500 platform was used to sequence the library, thereby generating a total of 260,117,346 raw reads. The transcription levels of 20 conserved miRNAs (including published reliable references miR171 [32], miR166b [31], miR159a [49], miR396a [50] and miR166b [51] were obtained and analyzed. Eight putative reference RNA targets (miR472, miR428b, miR396a, miR166b, miR3954, miR160, miR162-3p and miR403) with a median expression and the smallest standard deviations were selected from high-throughput sequencing data for identifying the most suitable normalizer for miRNA qPCR in citrus infected by Xcc.…”

“…Several studies have recently been performed for identifying appropriate reference genes for miRNA qPCR, of which the stem-loop method has been used more frequently. A set of miRNAs was selected for evaluation in plants, such as castor bean, cotton, watermelon, wheat and sugarcane [30][31][32][33]. Luo et al, 2014, found miR5059 and miR5072 were the best reference genes in different peach tissues under different abiotic stress treatments using poly(A)-tailed RT-qPCR approach [34].…”