RESUMO
Rapeseed (Brassica napus) is an allopolyploid hybrid (AACC genome) of turnip rape (B. rapa, genome: AA) and vegetable cabbage (B. oleraceae, genome: CC). Rapeseed oil is one of the main vegetable oils used worldwide for food and other technical purposes. Therefore, breeding companies worldwide are interested in developing rapeseed varieties with high yields and increased adaptation to harsh climatic conditions such as heat and prolonged drought. One approach to studying the mechanism of the epigenetically regulated stress response is to analyze the transcriptional changes it causes. In addition, comparing the expression of certain genes between stress- and non-stress-tolerant varieties will help guide breeding in the desired direction. Quantitative reverse transcription PCR (RT-qPCR) has been intensively used for gene expression analysis for several decades. However, the transfer of this method from model plants to crop species has several limitations due to the high accumulation of secondary metabolites, the higher water content in some tissues and therefore problems with their grinding and other factors. For allopolyploid rapeseed, the presence of two genomes, often with different levels of expression of homeologous genes, must also be considered. In this study, we describe the optimization of transcriptional RT-qPCR analysis of low-expression epigenetic genes in rapeseed, using Kinetochore Null2 (KNL2), a regulator of kinetochore complex assembly, as an example. We demonstrated that a combination of various factors, such as tissue homogenization and RNA extraction with TRIzol, synthesis of cDNA with gene-specific primers, and RT-qPCR in white plates, significantly increased the sensitivity of RT-qPCR for the detection of BnKNL2A and BnKNL2C gene expression.
RESUMO
Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence. Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) using the enteric pathogen Shigella flexneri serotype 5a strain M90T as model. However, this method can be employed in any other bacterial species of choice. In the first steps, total RNA is purified from bacterial cultures using the hot phenol method. Ribosomal RNA (rRNA) is specifically depleted via hybridization probes using a commercial kit. A 5'-monophosphate-dependent exonuclease (TEX)-treated RNA library enriched in primary transcripts is then prepared for comparison with a library that has not undergone TEX-treatment, followed by ligation of an RNA linker adaptor of known sequence allowing the determination of TSS with single nucleotide precision. Finally, the RNA is processed for Illumina sequencing library preparation and sequenced as purchased service. TSS are identified by in-house bioinformatic analysis. Our protocol is cost-effective as it minimizes the use of commercial kits and employs freely available software.