mRNA analysis plays a crucial role in understanding gene expression patterns and identifying potential drug targets and biomarkers for various diseases.
The analysis of mRNA involves several steps:
Isolation of RNA: RNA is isolated from the biological sample using various methods such as column-based purification, magnetic bead-based purification, or organic extraction.
Quantification and quality assessment of RNA: The concentration and purity of RNA are determined using spectrophotometry or fluorometry. The quality of RNA is assessed using gel electrophoresis or capillary electrophoresis.
cDNA synthesis: RNA is reverse-transcribed into complementary DNA (cDNA) using reverse transcriptase enzyme and oligo-dT primers.
PCR amplification: The cDNA is amplified using polymerase chain reaction (PCR) with gene-specific primers.
Gene expression analysis: The PCR products are analyzed for gene expression using various techniques such as real-time PCR, microarray analysis, or RNA sequencing.
Data analysis: The gene expression data is analyzed using various bioinformatics tools and software to identify differentially expressed genes, gene ontology, and pathway analysis.
To ensure the accuracy and reliability of the results, We take quality control in mRNA analysis, which is an essential step. The following are some of the quality control measures that are commonly used in mRNA analysis:
RNA quality assessment: The quality of RNA is assessed by measuring the RNA concentration, purity, and integrity using spectrophotometry, fluorometry, and gel electrophoresis. High-quality RNA with a high purity and integrity is essential for accurate mRNA analysis.
Reverse transcription quality control: The efficiency and accuracy of reverse transcription are checked by performing a reverse transcription control reaction with a known amount of RNA and comparing the resulting cDNA yield with the expected yield.
PCR quality control: The specificity and efficiency of PCR amplification are checked by performing a PCR control reaction with a known amount of template DNA and comparing the resulting amplicon yield with the expected yield.
Replicate analysis: Replicate analysis is performed to check the reproducibility of the results. Multiple replicates of the same sample are analyzed, and the results are compared to determine the level of variation.
Positive and negative controls: Positive and negative controls are included in the analysis to ensure the accuracy and reliability of the results. Positive controls contain known amounts of mRNA, while negative controls contain no mRNA.
ALL Chemistry Inc. supports mRNA product development, and we are experienced in designing and conducting strategic characterisation and quality control programs, including formulation and stability assessment. ALL Chemistry Inc. utilises high-end analytical instrumentation as well as a more traditional QC approach to generate validated analytical methods covering identity, quantity, purity, impurities (product and process related), and potency of the construct as well as of the delivery and container closure systems (extractables and leachables). At our laboratories, scientists test mRNA drug substances or drug products to fully characterise the material, as well as for quality control (QC) purposes to help you confidently assess batch-to-batch manufacture, process repeatability, and general quality of mRNA produced.
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