Validation of Exosomal Differentially-Expressed Genes
High-throughput sequencing and protein quantitative mass spectrometry are usually used to analyze the contents of exosomes. Among them, sequencing is an important means to find differentially expressed genes.Once differential genes have been identified, qualitative and quantitative validation of these differential genes is required to determine whether there are significant differences in the expression levels of these genes in exosomes derived from specific cells or tissues relative to the control group. Then, the functions of these differentially expressed genes are verified, such as knock-down, over-expression or functional recovery experiments, to establish the correlation between these gene expression changes and physiological or pathological state.
Real-Time Fluorescent Quantitative PCR (qPCR)
Real-time quantitative PCR (qPCR) technology, launched by ABI in 1996, has achieved a technological leap from qualitative to quantitative analysis. Compared with traditional PCR, qPCR not only provides higher specificity, but also effectively solves the problem of PCR contamination and has a high level of automation. These features make it an internationally recognized standard method for the quantitative analysis of nucleic acid molecules. Currently, the most commonly used methods for real-time quantification include dyes (e.g., SYBR Green I) and TaqMan probes. This technique has been widely used in many fields of molecular biology research, such as DNA and RNA quantification, single nucleotide polymorphism (SNP) analysis, genotype analysis, and RNA variant analysis.
Digital PCR (ddPCR)
Digital PCR technology enables the distribution of nucleic acid samples into a large number of independent and parallel micro-reaction units at the nanoliter scale. Within these units, some contain the target molecules (positive units), while others do not (negative units). Subsequently, PCR amplification is performed, and TaqMan chemical reagents or dye-labeled probes are used to detect the target sequences. This technique allows for the absolute quantification of target molecules and offers advantages such as high specificity, high sensitivity, precise absolute quantitation, good accuracy, and strong resistance to interference.
1.Red-Target and Green-Background (gDNA, CDNA; primers/probes; master mix)
2.Random distribution of molecules into partitions
3.Absolute quantification: Copies/ul
Luciferase Reporter Gene (Luc)
The Luciferase Reporter Gene (Luc) refers to a reporting system that uses luciferin as a substrate to detect the activity of firefly luciferase. Luciferase can catalyze the oxidation of luciferin to oxyluciferin, during which bioluminescence is emitted. This bioluminescence can be measured using a fluorometer, also known as a luminometer, or a liquid scintillation counter. The luciferin and luciferase-based bioluminescent system is extremely sensitive and efficient in detecting gene expression, making it an important method for studying the interaction between transcription factors and the promoter region DNA of target genes.
Services at NextGen Exsome
1.gPCR Exosomal Differentially Expressed Genes Validation
After reverse transcription of RNA, using real-time quantitative PCR (qPCR) validate the gene express.
Deliverables:
Report in DOCX format and machine raw data in PCRD format
2.ddPCR Exosomal Differentially Expressed Genes Validation
After reverse transcription of RNA, using digital PCR (dPCR) validate the gene express.
Deliverables:
Report in DOCX format and machine raw data in PCRD format
3.Luciferase Reporter (Luc) Gene Assay
Detect the regulatory relationships of miRNA with the 3’UTR of target mRNA, or with IncRNA/circRNA, or to examine the regulatory relationships of transcriptional regulatory factors with their target genes.
Deliverables:
Report in DOCX format and machine raw data in PZFX format