Transcriptomics

Transcriptomics is the analysis of the transcriptome, the collection of all ribonucleic acid (RNA) that is present in a sample (a cell, tissue or organ) at a given time. In humans, DNA segments are copied into RNA in a process called transcription, allowing a cell to act on the 'instructions' encoded in the DNA genome. Different types of RNA have different roles: messenger RNA (mRNA) is produced during gene expression as an intermediary between DNA and proteins, while other non-protein coding RNAs impact on a variety of cellular functions. A cell's transcriptome is constantly changing depending on its needs.

Transcriptomics can either refer to exploratory analysis of the entire transcriptome, primarily using RNA sequencing (RNA-seq), or to targeted analysis of known RNAs using techniques such as gene expression panels (GEPs).

Process

• RNA extraction
  Depending upon the experimental requirement either total RNA or mRNA is extracted from the tissues. Extracting RNA is a challenging task because it is weaker than DNA and thus can be fragmented easily either by external conditions or by the RNase. Also, sterile conditions must be strictly maintained during RNA extraction.
• Reverse transcription PCR
  Using the reverse transcription PCR method, a cDNA is constructed from the mRNA. The procedure of RT-qPCR is completed in the following steps: sample preparation, selection of primers, reaction preparation, RT-PCR cyclic condition and strand synthesis.

Fig.1 The process of transcriptomics analysis: in vivo mRNA synthesis, in vitro cDNA construction and in silico data analysis.

Analysis

Microarray and RNA - seq are two methods used for transcriptomics analysis.

• Microarray
  The microarray is a special type of set up in which the probes are immobilised on the solid glass surface.

• The sample (which is cDNA reverse transcribed from the mRNA) is fragmented using restriction digestion method and labelled with the fluorochrome.
• The sample, as well as the control - DNA or cDNA, is prepared with the same procedure and allows to hybridize on the glass slide.
• From the millions of immobilised probes, the cDNA fragment finds its complementary sequence and hybridized with it.
• The microarray scanner scans the entire slide and collects the data of hybridization. Based on the hybridization signal, the amount of gene expression can be determined.

• RNA - seq
  The RNA sequencing is a high-throughput method which quantifies the RNA present in a sample and also captures the sequence data.

• The RNA is first reverse-transcribed into the cDNA and fragment library is constructed using the restriction digestion method.
• For here, in RNA sequencing typically 100 to 1000 or 10000 bp fragments are created and allow for sequencing.
• The mechanism of sequencing is the same as the DNA sequence. The fragments are read in the machines and aligned based on the sequence information available with us. The "contings" are generated, aligned and computationally analysed.

Applications

• Cancer
  GEP tests which examine a subset of RNAs have shown promise in understanding prognosis and guiding treatment decisions in cancer and are already used for this purpose in breast cancer.
• Rare disease
  Clinical research studies are examining the utility of performing transcriptome analysis alongside whole genome or exome sequencing (WGS / WES) in rare disease. Transcriptomics is showing promise in improving the number of diagnoses made.
• Infectious disease
  Analysis of gene expression patterns in patients is being investigated as a novel tool to assist in the diagnosis and management of infectious disease, for example distinguishing between bacterial and viral infections, and predicting the severity of sepsis.

Related Products

Microarray System

Microarray scanners are widely used in functional biology, cellomics, gene expression and drug discovery research. When buying a microarray scanner, important features to consider are sensitivity, resolution, scan area, rapidity of scan, and ease of use. Microarray Scanners usually have red, green, and blue excitation wavelengths and a wide choice of up to 8 emission filters that enable imaging of an extensive variety of fluorophores.

Pipette

Pipette is a small piece of apparatus which typically consists of a narrow tube into which fluid is drawn by suction (as for dispensing or measurement) and retained by closing the upper end. Pipettes are a common laboratory tool and come in a multitude of channels, displacements and construction materials. There are different pipettes for all kinds of uses within the lab.

Nucleic Acid Extraction and Purification Apparatus

Nucleic acid extraction instrument, also known as nucleic acid purification instrument, is an instrument that automatically completes the extraction of sample nucleic acid by using the matching nucleic acid extraction reagent. Magnetic bead automatic nucleic acid extraction and purification system uses magnetic bead method technology, which can be widely used in genomics, disease control medical treatment, food safety, forensic identification, etc.

Digital PCR System (dPCR)

Digital PCR Systems (dPCR) is incredibly useful in applications such as mutation detection, copy number variation, rare sequence detection, gene expression analysis, miRNA analysis and next generation sequencing sample quantification. It provides a precise measure of DNA molecules in each drop. In some cases, Digital PCR Systems (dPCR) must be used in combination with a 96-deep well reaction module.

Electrophoresis System

Electrophoresis equipment applies an electric charge to molecules, causing them to migrate towards their oppositely charged electrode. The technique is found in all research and clinical laboratories utilizing DNA and protein applications, and is divided into gel and capillary techniques. Choice of electrophoresis equipment depends on the molecule of study, appropriate method of separation and downstream application.

Centrifuge

A centrifuge is any device that applies a sustained centrifugal force—that is, a force due to rotation. The widest use of centrifuges is for the concentration and purification of materials in suspension or dissolved in fluids. Suspended particles denser than the suspending liquid tend to migrate toward the periphery, while those less dense move toward the centre.

STEMart provides you with a variety of transcriptomics equipment or consumables to meet your various R&D and application needs. If you have any questions or requirements for techniques for the study of gene function, please feel free to contact us.