Nonsense-Mediated mRNA Decay (NMD) Analysis

Nonsense-mediated mRNA decay (NMD) is a mRNA quality monitoring mechanism that exists widely in eukaryotic cells. CD Genomics provides NMD analysis services based on qPCR technology for further elucidation of the mechanism of gene expression.

Overview

The degradation of mRNA is a very important regulation approach in the process of gene expression, among which nonsense-mediated mRNA decay is a highly conserved RNA monitoring mechanism that is widely found in eukaryotes recently. This mechanism destroys mRNA containing premature termination codons, which can prevent the production of abnormal truncated proteins and ensure the normal physiological function of cells. It plays an important role in human genetic diseases and cancer etiology.

CD Genomics provides nonsense-mediated mRNA decay analysis service to verify whether the mRNA level degradation is due to the occurrence of NMD by the following 3 steps: First, construct a eukaryotic expression vector: clone the target genome fragment with a mutation site (producing a premature termination codon, PTC), and construct a recombinant expression vector. Secondly, the gene expression level was detected: after the recombinant vector was transfected into the cells, the expression differences caused by the mutation were detected from the mRNA level (qPCR). Finally, NMD validation: if the detection indicates that mRNA level degradation occurs after mutation, the possibility of NMD degradation can be further verified.

Features

One-Stop Service Customized Service Experienced Scientist Team Professional Bioinformatics
Design and implement the whole experiment scheme. Different methods can be selected for NMD validation to increase the possibility of obtaining positive results. Can provide a full set of professional services from experimental design, sample testing, data analysis, etc. Strong bioinformatic team provides conventional analysis and in-depth data analysis.

Project Workflow

Sample Preparation

1. Vector Construction

Cloning gene fragment with PTC and construction of expression vector.

Library Preparation

2. Gene Expression Assay

qPCR for the detection of expression differences caused by mRNA mutations.

Sequencing

3. NMD Vadidation

Further verification of the NMD degradation possibility.

Data Analysis

4. Data Analysis

Preprocess and visualize results, and perform custom bioinformatics analysis.

In-depth data analysis:

  • Predict the possibility of NMD
  • Design NMD experiment program fishing
  • Gene was extracted and eukaryotic expression vector was constructed
  • Gene expression analysis
  • NMD degradation detection

Sample Requirements

Tissue sample: above 100 mg

Cell sample: cell amount: above 1×107

RNA sample: RNA quantity ≥ 5 μg; RNA purity: OD260/280 = 1.6~2.3; OD260/230 ≥ 1.5; RNA quality: 28S:18S ≥ 1.5 or RIN ≥ 7

Please make sure that the RNA is not significantly degraded.

Sample storage: Cell samples or fresh tissue pieces (cut into 5-10 mg pieces) can be treated with TRIzol or RNA protectant, frozen in liquid nitrogen, and stored at -80°C. RNA samples can be dissolved in ethanol or RNA-free ultrapure water, and stored at -80°C. Avoid repeated freezing and thawing during sample storage.

Shipping Method: When shipping RNA samples, the RNA sample is stored in a 1.5 mL Eppendorf tube, sealed with sealing film. Shipments are generally recommended to contain 5-10 pounds of dry ice per 24 hours.

Deliverable:

1. Constructed expression vector;

2. Service report: including bioinformatics prediction results, vector construction reports, gene expression level detection results, NMD detection results.

References:

  1. Supek F, Lehner B, Lindeboom RG. To NMD or Not To NMD: nonsense-mediated mRNA decay in cancer and other genetic diseases. Trends in Genetics. 2021 Jul 1;37(7).
  2. Pereverzev AP, Gurskaya NG, Ermakova GV, et al. Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Scientific reports. 2015 Jan 12;5(1).
* For Research Use Only. Not for use in diagnostic procedures.


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