Introduction

Small interfering RNA (siRNA) is a widely used molecular biology tool for gene silencing studies through the RNA interference (RNAi) pathway. Chemically synthesized siRNAs can specifically reduce target gene expression by promoting sequence-specific degradation of complementary mRNA.

MiaoLing siRNA products are ready-to-use double-stranded RNA reagents designed for gene knockdown experiments. This guide provides product information, handling instructions, transfection procedures, and recommended controls for successful siRNA experiments.

I. Product Information

MiaoLing chemically synthesized siRNA products are 21–23 nucleotide (nt) double-stranded small interfering RNA molecules supplied as ready-to-use reagents.

The products are shipped at room temperature in a lyophilized (freeze-dried) powder format.

Upon receipt:

• Store siRNA products at −20°C.
• Each tube contains either 5 nmol or 2.5 nmol siRNA.
• Conversion reference:

1 OD = 2.5 nmol = 40 μg

II. siRNA Handling and Preparation

1. siRNA Dissolution

siRNA is supplied as a thin, dry film attached to the inner wall of the tube.

Before opening:

1. Briefly centrifuge the tube to collect the siRNA powder at the bottom.
2. Slowly open the tube to avoid sample loss.
3. Add sufficient RNase-free water or sterile ddH₂O.
4. Close the tube and gently vortex until completely dissolved.

For products containing 2.5 nmol siRNA, dissolve with:

125 μL RNase-free water

to prepare the recommended stock solution.

2. RNA Handling Precautions

To prevent degradation caused by external factors, including RNases, extreme pH conditions, or temperature fluctuations, all procedures should follow standard RNase-free operating practices.

Recommended handling conditions:

• Keep siRNA samples on ice during experiments whenever possible.
• After use, store siRNA carefully at −20°C to −80°C.

Avoid repeated freeze–thaw cycles to maintain siRNA stability.

III. siRNA Transfection Protocol

The following protocol is provided as a reference example using:

• 24-well cell culture plates
• Final siRNA concentration: 50 nM

Optimization experiments should be performed to determine the optimal siRNA concentration, cell density, and transfection conditions for each cell type.

1. Cell Seeding

A. Adherent Cells

Approximately 24 hours before transfection, seed:

0.5–2 × 10⁵ cells/well

in 400 μL antibiotic-free culture medium.

At the time of transfection, the recommended cell confluency is:

30–50%

B. Suspension Cells

Approximately 24 hours before transfection, seed:

0.5–2 × 10⁵ cells/well

in 400 μL antibiotic-free culture medium.

At the time of transfection, the recommended cell density is:

4–8 × 10⁵ cells/well

2. siRNA Transfection Procedure

Step 1: siRNA Dilution

Dilute siRNA using 50 μL Opti-MEM™ medium.

Adjust the siRNA concentration to achieve a final concentration of:

50 nM in the transfection system

Mix gently by pipetting 3–5 times.

Step 2: Transfection Reagent Preparation

Gently invert the transfection reagent before use.

Dilute:

1 μL transfection reagent

with:

50 μL Opti-MEM™ medium

Mix gently by pipetting 3–5 times.

Incubate at room temperature for 5 min.

Step 3: Formation of siRNA Transfection Complexes

Combine the diluted siRNA solution with the diluted transfection reagent.

Mix gently by pipetting 3–5 times.

Incubate at room temperature for:

20 min

to allow formation of siRNA transfection complexes.

Step 4: Addition to Cells

Add the siRNA-transfection reagent complexes into each well of the 24-well plate.

Recommended volume:

100 μL per well

Gently rock the plate back and forth to distribute the complexes evenly.

Step 5: Cell Incubation

Incubate cells at:

• 37°C
• 5% CO₂

for:

18–48 hours

After 4–6 hours of transfection, replace the medium with fresh complete culture medium if required.

IV. Recommended Experimental Controls

1. Negative Control siRNA

Negative control siRNA does not target any known gene and should not induce gene silencing.

It is used to determine whether observed changes in gene expression are caused specifically by sequence-dependent RNA interference rather than nonspecific experimental effects.

2. FAM-Labeled Negative Control siRNA

FAM-labeled negative control siRNA contains the fluorescent dye FAM (fluorescein amidite).

It is commonly used to evaluate:

• Transfection efficiency
• Cellular uptake of siRNA
• Optimization of transfection conditions

3. Positive Control siRNA

Positive control siRNA targets the housekeeping gene GAPDH and has validated gene silencing activity.

It is used to confirm the reliability of:

• siRNA transfection procedures
• RNA extraction methods
• Gene expression detection assays

V. Frequently Asked Questions

What is siRNA used for?

siRNA is widely used for sequence-specific gene knockdown studies by reducing target mRNA levels through RNA interference.

How should siRNA be stored?

Lyophilized siRNA should be stored at −20°C after receipt. Reconstituted siRNA solutions should be stored at −20°C to −80°C depending on experimental requirements.

Why are negative and positive controls important in siRNA experiments?

Controls help distinguish specific gene silencing effects from nonspecific effects and verify that transfection and detection procedures are working properly.

Related Products

MiaoLingPlasmid provides:

• siRNA Products
• Gene Knockdown Solutions
• Plasmid-Based RNAi Tools
• Expression Plasmids
• Custom Molecular Biology Services