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RNA Sequencing

 

Introduction

This method is also called as Metatranscriptomic sequencing or Metatranscriptomics, goes one step ahead of shotgun metagenomics and whole genome sequencing by looking at the RNA sample instead of DNA. The transcriptome is the complete set of transcripts in a cell, and their quantity, for a specific developmental stage or physiological condition. Understanding the transcriptome is essential for interpreting the functional elements of the genome and revealing the molecular constituents of cells and tissues, and also for understanding development and disease. The key aims of transcriptomics are: to catalogue all species of transcript, including mRNAs, non-coding RNAs and small RNAs; to determine the transcriptional structure of genes, in terms of their start sites, 5′ and 3′ ends, splicing patterns and other post-transcriptional modifications; and to quantify the changing expression levels of each transcript during development and under different conditions

 

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Applications

•Global view of gene activity

•Novel gene and splice variant discovery

•Chromosomal rearrangement analysis

•Whole mRNA transcript expression analysis

 

Data Analysis

Primary Analysis

•Assembly and Mapping statistics

•Gene Expression profile

•SNPs and InDels calling

 

Secondary Analysis

•Differentially expressed genes (DEGs)

•Gene Annotations and Ontology Analysis

•Customised Analysis

Library Type: Eukaryotic RNA-Seq(cDNA library)

RNA Integrity Number(Agilent 2100)

Amount ≥ 6.8 (Animal), with smooth base line

Amount ≥ 6.3 (Plant and Fungus), with smooth base line

Purity (NanoDrop)

OD 260/280 = 1.8 - 2.2;
OD 260/230 ≥ 1.8;

Total RNA

Amount ≥ 0.4 μg

Total RNA (Blood)

Amount ≥ 0.8 μg

Total RNA (Single Cell)

Amount ≥ 100 ng

 

Amplified cDNA (double-stranded)

Amount ≥ 100 ng

Fragments between 400bp and 5000bp with main peak at ~2000bp

Purity (NanoDrop)

OD 260/280 = 1.8 - 2.0;
OD 260/230 ≥ 1.8;

 

Eukaryotic RNA-Seq (strand specific library)

Total RNA

Amount ≥ 0.8 μg

Amount ≥ 6.8 (Animal), with smooth base line
Amount ≥ 6.3 (Plant and Fungus), with smooth base line

Purity (NanoDrop)

OD 260/280 = 1.8 - 2.2;
OD 260/230 ≥ 1.8;