As Exon 8 corresponds to catalytic Lag1p domain, overexpression of AS transcript of CERS2 in Luminal B cancer cells leads to a reduction in the level of very-long-chain ceramides compared to overexpression of protein-coding (PC) transcript of CERS2. Differential AS-based survival analysis shows that this AS event of CERS2 is a poor prognostic factor for Luminal B patients. We validated this exon 8 skipping event in Luminal B cancer cells compared to normal epithelial cells, and in patient-derived tumor tissues compared to matched normal tissues. We show that ceramide synthase 2 ( CERS2 ) undergoes a unique cassette exon event specifically in Luminal B subtype tumors. Here, we present specific signature of alternative splicing (AS) events of sphingolipid genes for each breast cancer subtype from the TCGA-BRCA dataset. Global dysregulation of RNA splicing and imbalanced sphingolipid metabolism has emerged as promoters of cancer cell transformation.
However, RT-PCR appears to be more sensitive than RNA-Seq for detecting changes in existing isoform abundance In conclusion, RNA-Seq can be used to identify splicing abnormalities caused by splicing mutations, but still, further improvement must be made on the data analysis pipeline. For RNA-Seq, although the FPKM differences between control and mutated samples were not significant, using Sashimi plots function and psi calculation showed evidence for abnormal splicing events in the mutated samples. However, Sanger sequencing results were not informative enough due to their lack of sequence data. Data analysis was dealt with using BaseSpace and IGV. An Illumina Nextseq system was used for RNA-Seq. After RT-PCR was conducted and viewed on an agarose gel under UV, the bands were cut out and sent for Sanger sequencing. 20 RNA samples extracted from immortalised lymphoblastoid cell lines (10 controls, 8 samples with individual mutations in BRCA1 and BRCA2, and 2 samples that were either treated or untreated with cycloheximide) were analysed using two methods reverse transcription PCR (RT-PCR) and RNA sequencing (RNA-Seq). The aim of this project was to investigate the utility of RNA sequencing for the detection and characterisation of splicing abnormalities in lymphoblastoid cell lines harbouring variants within the BRCA1 and BRCA2 tumour suppressor genes. One of the applications is called RNA sequencing which is well known for its ability for deep and broad coverage sequencing to study RNA. High throughput sequencing applications have become essential in genome and transcriptome analysis nowadays. Some breast cancer cases are caused due to genetic changes in the BRCA1 and BRCA2 genes.
HOW TO FIND SERUM SERIAL NUMBER SPLICE SERIES
In addition, we validate the use of an absolute standardĬurve based on a dilution series of fluorometrically quantified PCR products.īreast cancer is the most common type of cancer in women worldwide and is the second leading cause of cancer death. We describe here a novel method for creating a reliable standardĬurve using one plasmid containing both alternative transcripts. Primer to quantify isoforms that differ greatly in abundance. We propose the use of a boundary-spanning Overview of three different possibilities for detecting alternative transcripts is given. We use skipping of exon 37 in the NF1 gene as a model to compare and evaluate the different strategies for quantitating splice variants using real-time PCR. To study its application in quantification of splice isoforms. The many advantages of real-time PCR have made this technique attractive To the intrinsic limitations of conventional methods. So far, accurate quantification of splice variants has been laborious and difficult due A reliable and robust method for measuring the expression of alternatively spliced transcripts is an important step in investigating