siRNA Interference Detection Services

RNA interference (RNAi) is an experimental technique in which a small (19 -23bp) exogenous double-stranded RNA (siRNA) homologous to the coding region of an endogenous mRNA is introduced into a cell, triggering efficient and specific degradation of the corresponding mRNA in the cell, resulting in the silencing of specific gene expression (knock down). BOC Sciences offers a service to test the effects of RNAi interference. We use real-time quantitative PCR to detect knock down effects at the mRNA level and Western blot to detect knock down effects at the protein level.

Introduction

Gene silencing is an important form of gene expression regulation. It is mainly manifested as down-regulation of the expression of a specific gene or inhibition of expression. There are two main aspects of gene silencing: gene silencing at the transcriptional level and gene silencing at the post-transcriptional level. The former is mainly caused by gene methylation, heterochromatinization and positional effects. The latter is caused by antisense RNA and RNA interference. Among them, RNA interference technology has shown great application prospects in gene function research and drug development in the post-genome era by efficient and specific target gene inhibition.

It is a phenomenon of silencing of homologous gene expression induced by double-stranded RNA. In RNA interference experiments, the selection of efficient siRNAs is critical for successful silencing or shutting down of gene expression as the efficiency of gene silencing mediated by siRNAs is unpredictable. Both the detection of the silencing effect of specific siRNAs and the selection of efficient siRNAs can be verified by bio-experimental means of real-time quantitative PCR or Western blot.

Factors influencing the efficiency of siRNA interference include:

• shRNA vector components-e.g. loop loops, thermodynamic stability of shRNAs.
• Target gene properties-e.g. target gene secondary structure, target gene microenvironment.
• Experimental system-e.g. target cells (primary cells), transfection efficiency will be lower. Control of transfection time, cells in a healthy state are conducive to higher transfection efficiency. Control of observation time, which depends on the half-life of the target protein. Transfection reagents and transfection steps, which can also affect transfection efficiency.
• The off-target nature of shRNA.
• The existence of a compensatory pathway for protein expression in organisms.

Experimental Process

• Selection of siRNA Expression Vectors.
• Designing siRNA Target Sequences.
• Based on the target mRNA sequence submitted by the client, design 3 RNA interference target sequences, the target sequence is generally 19-21nt long.
• For each selected siRNA target sequence, the siRNA sense and antisense strands are designed to be linked in a loop (9nt or 10nt) to form a stable hairpin RNA, called shRNA (short hairpin RNA).
• Synthesis templates.
• Extraction of positive clone plasmids.
• Transfected cells. The plasmid is directly transfected or transfected by virus packaging, and the positive identification of the transfected cells and the cultivation of the transfected cell lines are carried out.
• Detection of interference effects.
• Gene-level assays RNA extraction, reverse transcription, realtime PCR to detect interference.
• Protein level assay Western Blot to detect protein level interference.

Service Description

• For the siRNA service, the customer needs to discuss the experimental plan with us, and provide the target gene name, sequence or GeneBank ID, and the name of the vector to be inserted.
• As the inserted fragments in the constructed siRNA expression vector can form a hairpin structure, this may affect sequencing. PCR products obtained from plasmid amplification will be sequenced if we have unsatisfactory plasmid sequencing results.

Provide Results

The experimental report includes a siRNA sequence, a sequencing report of constructed vector, a fluorescent quantitative PCR detection report, Western Blot pictures, all experimental data and detailed experimental steps.