Protein analysis
Activity-based protein profiling (analyzing lysates)
Labeled lysate with the FP-ABP, added a biotin tag by click chemistry
Purified enzymes through streptavidin affinity chromatography
Washed and performed on-resin trypsin digest
Analyzed by high-resolution LC-MS
Genotyping using commercial Line Probe Assay (Innogenetics) , confirmed by sequencing of Core PCR products followed by phylogenetic analysis
Microarray
RNA sequencing
RNA sequencing (high-throughput cDNA sequencing or RNAseq) uses the NGS technology for discovering novel RNA sequences, and quantifying all transcripts in a cell.
In contrast to microarrays, RNAseq offers a broader dynamic range, which makes this platform more sensitive in the detection of transcripts with low abundance.
Current instruments generate more than 500 gigabases in a single run
Input files: Illumina FASTQ reads, SOLiD, Solexa data
Protocol begins with raw sequencing reads and produces a transcriptome assembly
RNAseq analyses steps
Pre-processing
Quality control (excluded reads with a score Q < 20 )
Read mapping (alignment)
Differential expression (DE) analysis
Single nucleotide polymorphism (SNP) analysis
Gene isoform and splicing variant detection
Networks and function
* Information on gene network inference methods, miRNA-target predictions, integration of protein-protein interactions, alternatively splicing variants or promoter enriched sites
RNA Integrity Number (RIN) is the most widely used approach to assess in vitro RNA degradation
RIN relies on 18S:28S ratio
RNA-seq-based, gene expression profiling studies
transcriptome of stem cell derived antibiotic selected cardiac bodies
Tools to analyze RNAseq data: C, Python or R
TopHat and Cufflinks are tools for gene discovery and comprehensive expression analysis of RNA-seq data
CummeRbund: tool for visualizing RNA-seq analysis results
Trinity,Trans-Abyss,Oases: Gene discovery sfollowing de novo transcriptome assembly
Activity-based protein profiling (analyzing lysates)
Labeled lysate with the FP-ABP, added a biotin tag by click chemistry
Purified enzymes through streptavidin affinity chromatography
Washed and performed on-resin trypsin digest
Analyzed by high-resolution LC-MS
Genotyping using commercial Line Probe Assay (Innogenetics) , confirmed by sequencing of Core PCR products followed by phylogenetic analysis
Microarray
A DNA microarray (DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. These are used to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome i.e parallel analysis of genetic material in the genome
Uses hybridization to monitor DNA/RNA abundance on a genome.
Cell -----> extract RNA -----> reverse transcription (purification and labelling by IVT)----->fragmenattion (heat, Mg++) ----->hybridization-----> laser scanning ----->gene expression-----> ratio analysis
miRNAs plays role in normal physiology, also diseases like cancer. Hybridization based microarray technology has been used for miRNA profiling, but is hindered by its narrow detection range, more susceptibility to technical variation, and lack of ability to characterize novel miRNAs and sequence variation.
RNA sequencing is better than microararysRNA sequencing
RNA sequencing (high-throughput cDNA sequencing or RNAseq) uses the NGS technology for discovering novel RNA sequences, and quantifying all transcripts in a cell.
In contrast to microarrays, RNAseq offers a broader dynamic range, which makes this platform more sensitive in the detection of transcripts with low abundance.
Current instruments generate more than 500 gigabases in a single run
Input files: Illumina FASTQ reads, SOLiD, Solexa data
Protocol begins with raw sequencing reads and produces a transcriptome assembly
RNAseq analyses steps
Pre-processing
Quality control (excluded reads with a score Q < 20 )
Read mapping (alignment)
Differential expression (DE) analysis
Single nucleotide polymorphism (SNP) analysis
Gene isoform and splicing variant detection
Networks and function
* Information on gene network inference methods, miRNA-target predictions, integration of protein-protein interactions, alternatively splicing variants or promoter enriched sites
RNA Integrity Number (RIN) is the most widely used approach to assess in vitro RNA degradation
RIN relies on 18S:28S ratio
RNA-seq-based, gene expression profiling studies
transcriptome of stem cell derived antibiotic selected cardiac bodies
Tools to analyze RNAseq data: C, Python or R
TopHat and Cufflinks are tools for gene discovery and comprehensive expression analysis of RNA-seq data
TopHat: Aligns reads to the genome and discovers transcript splice (based on Bowtie)
Cufflinks: Uses this map against the genome to assemble the reads into transcripts. It clusters transcripts such as that share the same transcription start site (TSS)
CummeRbund: tool for visualizing RNA-seq analysis results
Trinity,Trans-Abyss,Oases: Gene discovery sfollowing de novo transcriptome assembly
CAP-miRSeq: A comprehensive analysis pipeline for deep microRNA sequencingcDNA sequencing with Sanger sequencers drastically expanded our catalog of known human genes.
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