Wednesday, February 3, 2016

Bio (1): Proteomics; Microarray and Transcriptomics...

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
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 microararys
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
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: CPython 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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