Cell transfection reagents have become routine reagents for studying and controlling gene function in eukaryotic cells. Transfection reagents are widely used in gene function research, gene expression regulation, and mutation analysis, as well as gene therapy, cell therapy, protein production, and vaccine production. So what is transfection? And how to choose a kind of transfection reagent based on your experiments?

 

What are the types of transfection?

The features of transfection reagents from Yeasen

How to choose a kind of transfection reagent based on your experiments?

Application case

Reference for transfection conditions

FAQs

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What are the types of transfection?

According to whether the nucleic acid is integrated into the host cell chromosome after transfection, it is divided into "transient" (transient transfection) and "stable" (stable transfection). The transfection efficiency, cytotoxicity, effects on normal physiology, and gene expression levels of different transfection methods are different. The principles, applications, and characteristics are compared in the following table:

Table 1 Comparison of different transfection methods

Technology	Principles	Advantages	Disadvantages
Chemical transfection method
Cationic liposomes	Positively charged liposomes form complexes with negatively charged phosphate groups of nucleic acids and are endocytosed by cells.	Quick and easy operation Results are reproducible High transfection efficiency Transfectable DNA, RNA, and protein Suitable for the production of transient and stable proteins Can be used for in vivo transfection	Requires optimization of conditions (some cell lines are sensitive to cationic liposomes) Some cell lines are not easily transfected The presence of serum interferes with complex formation, resulting in low transfection efficiency Depletion of serum in the medium increases cytotoxicity
Calcium Phosphate Coprecipitation	Calcium phosphate DNA complexes adsorb to cell membranes and are endocytosed by cells	Cheap and easy to obtain Suitable for the production of transient and stable proteins High transfection efficiency (not limited to cell lines)	Requires careful reagent preparation – CaPO4 solutions are sensitive to changes in pH, temperature, and buffer salt concentration poor repeatability Cytotoxic, especially against primary cells RPMI medium cannot be used due to its high concentration of phosphate Not suitable for transfection in animals.
Dextran	The complex formed by the interaction of the positively charged DEAE-dextran and the negatively charged phosphate backbone of the nucleic acid is endocytosed by the cell.	Easy to use Results are reproducible Cheap	Chemically toxic to certain cells For transient transfection only Low transfection efficiency, especially in primary cells.
Other cationic polymers	The positively charged polymer forms a positively charged complex with the negatively charged phosphate group of the nucleic acid, then interacts with the negatively charged proteoglycan on the cell surface, and enters the cell through endocytosis.	Stable in serum, insensitive to temperature High transfection efficiency (limited cell lines) Results are reproducible	Toxic to some cells Not biodegradable (dendrimer) Mostly used for transient transfection, less used for stable transfection
Biotransfection method
Viral transfection	Instinct infects cells and delivers genetic material	High transfection efficiency For difficult-to-transfect cell lines Can be used for in vivo transfection Can be used to construct stable or transient expression cell lines	Transfected cell lines must contain viral receptors Gene insertion size is limited (~10 kb for viral vectors, ~100 kb for non-viral vectors) Technically difficult and time-consuming to construct recombinant proteins There are biosafety issues (activation of underlying disease, immunogenic response, cytotoxicity, insertional mutagenesis, malignant transformation of cells)
Physical transfection method
Electric transfer	The high pulse voltage disrupts the cell membrane potential, and the DNA is introduced through the pores formed in the membrane.	The principle is simple Optimized conditions to produce reproducible results no carrier required Unlimited cell types and conditions Optimized conditions for rapid transfection of large numbers of cells	Special equipment required Electro-to-pulse and voltage parameters need to be optimized Great damage to cells The cell death rate is high and therefore requires a large number of cells Irreversibly damages cell membranes, lysing cells
Biotransmission Particle Delivery (Particle Bombardment)	The DNA is precipitated with microscopic heavy metal particles, and then the coated particles are projected into the cells with a ballistic device, and the DNA is gradually released and expressed in the cells.	Unlimited cell types and conditions Can be used for transfection in animals Direct method and reliable results Unlimited size and number of imported genes	Requires expensive equipment physical damage to the sample The cell death rate is high and therefore requires a large number of cells Need to prepare particles Relatively low transfection efficiency Expensive for research applications
Microinjection	Micromanipulation is used to inject DNA directly into the nucleus of the target cell.	Unlimited cell types and conditions single cell transfection Direct method and reliable results Unlimited size and number of imported genes No carrier required	Requires expensive equipment Technically demanding and labor-intensive (only one cell can be transfected at a time) A limited number of transfected cells Often causes cell death

 

The features of transfection reagents from Yeasen

For DNA transfection reagents and RNA transfection reagents, Yeasen Biotechnology has a strong R&D and production team, continuously optimizes formulas, improves production processes, and has launched a variety of products based on cationic liposomes and cationic polymers. Scientific research institutions and enterprises provide a full range of products, and the product line covers all fields involved in transfection reagents.

Hieff Trans™ Liposomal Transfection Reagent	Hieff Trans™ Suspension Cell-Free Liposomal Transfection Reagent
40802ES	40805ES
Hieff Trans™ in vitro siRNA/miRNA Transfection Reagent	Polyethylenimine Linear(PEI) MW40000（rapid lysis）
40806ES	40816ES

• High efficiency: suitable for transient transfection or stable transfection of cell lines.
• Low toxicity: Transfected cells remain well-viable.
• Wide adaptability: comprehensive coverage of common cells and difficult-to-transfect primary cells.
• Easy to operate: suitable for medium in the presence of serum, without changing the medium before and after transfection.
• Cost-effective: economical and practical, high transfection efficiency, low price.

How to choose a kind of transfection reagent based on your experiments?

The selection of transfection reagents needs to be selected according to the different experimental purposes and experimental contents, such as the transfected substances, specific cells, convenience of operation and other factors.

Product	Hieff Trans™ Liposomal Transfection Reagent	Hieff Trans™ Suspension Cell-Free Liposomal Transfection Reagent	Hieff Trans™ in vitro siRNA/miRNA Transfection Reagent	Polyethylenimine Linear(PEI) MW40000（rapid lysis）
Cell type	conventional cell	conventional cell	conventional cell	conventional cell
	/	/	difficult-to-transfect cells	difficult-to-transfect cells
Nucleic acid type	DNA	DNA	/	DNA
	siRNA	siRNA	siRNA	/
	/	/	miRNA	/
	/	/	mimic miRNA	/
	/	/	antimiRNA	/
	DNA/siRNA co-transfection	DNA/siRNA co-transfection	/	/
	virus packaging	virus packaging	/	virus packaging

 

Application case

Hieff Trans™ Liposomal Transfection Reagent

Hieff Trans™ is supplied in sterile liquid form. Generally, for 24-well plate transfection, about 1.5 μL each time, 1 mL of Hieff Trans™ can do about 660 transfections; for 6-well plate, about 6 μL each time, 1 mL of Hieff Trans™ can do about 660 transfections. 160 transfections;

For more details, please see Confidence in transfection with Hieff Trans™ Lipofectamine Reagent

 

Polyethylenimine Linear (PEI) MW40000 (rapid lysis)

PEI 40000 is a highly charged cationic polymer with a molecular weight of 40,000 that binds negatively charged nucleic acid molecules very easily, forming a complex and allowing the complex to enter cells. PEI 40000 is a transient transfection reagent with low cytotoxicity, high transfection efficiency, and high gene expression efficiency in cells such as HEK293 and CHO. Linear PEI transfection reagents have been validated for a wide range of cell lines including HEK-293, HEK293T, CHO-K1, COS-1, COS-7, NIH/3T3, Sf9, HepG2, and Hela cells. The transfection efficiency is as high as 80%~90%.

 

For more details, please see New favorite for transfection —— Linear PEI MW 40000, a more efficient transfection reagent

 

Hieff Trans™ in vitro siRNA/miRNA Transfection Reagent

This product can achieve over 90% expression efficiency of 1 nM siRNA in a wide range of cell lines, avoiding off-target effects. Suitable for transfection of a variety of cells, including Hela, MCF-7, HepG2, CHO and other adherent cells; and difficult-to-transfect suspension cell lines, such as K562 or THP-1 cells, can achieve 80% silencing efficiency; Including some primary cells, primary human fibroblasts and primary human hepatocytes, etc., the silencing efficiency of 80% can be achieved.

 

Reference for transfection conditions

In addition to the instructions for each product, customers operate according to their specific experimental content, and there will be different differences in the amount of use. According to the in vitro cell transfection conditions reported by customers after using the product, they have been sorted out for your reference.

Product Name/Item Number	Hieff Trans™ Liposomal Transfection Reagent/40802ES
Cell	Culture vessel	Cell plating densities	DNA	Hieff trans	Transfection efficiencies
A549	6 well	90%	0.7 μg	1.15 μL	+++
BV 2	24 well	95%	0.2 μg	0.2 μL	++
C2C12	24 well	80% - 90%	1 μg	5 μL	++
DF 1	24 well	80% - 90%	0.5 μg	0.5 μL	+++
H520	6 well	80%	1.2μg	6 μL	++
HaCaT	96 well	70%	100 ng	1 μL	++
HCT116	6 well	90%	4 μg	10 μL	++
HEK 293	6 well	95%	2 μg	10 μL	80 - 90%
HEK 293FT	24 well	85%	1 μg	4 μL	90%
HEK 293T	12 well	1×105	1 μg	2 μL	+++
HEK 293T（suspension）	30 ml	80%	30 μg	60 μL	++
Hela	12 well	90%	0.2μg	0.6 μL	90%
Hela	12 well	80%	1 μg	3 μL	+++
HepG2	12 well	80%	1 μg	3 μL	++
HUVEC	24 well	80%	1 μg	2 μL	++
MCF10A	10 cm dish	60%	5 μg	15 μL	++
N2A	24 well	70% - 80%	300 ng	900 μL	+
NCI H1975	6 well	80%	4 μg	10 μL	+++
NIH 3T3	6 well	90%	4 μg	10 μL	+++
Raw 264.7	35 mm dish	80%	1 μg	2 μL	90%
Vero	6 well	80%	3 μg	9 μL	+++
Cell	Culture vessel	Cell plating densities	siRNA	Hieff trans	Transfection efficiencies
HK2	6 well	65%	100 pmol	6 μL	+++

 

FAQs

1 Hieff Trans™ Liposomal Transfection Reagent

1.1 Q: Can serum be present when preparing nucleic acid transfection reagent complexes?

A: The presence of serum will affect the formation of liposomes. It is recommended to use a serum-free medium (generally MEM medium) when preparing nucleic acid transfection reagent complexes.

1.2 Q: What should I pay attention to when using Hieff Trans™ Liposomal Nucleic Acid Transfection Reagent?

A:

1) When the cells are transfected, the cell density is preferably 80%-95%, and the specific plating density is determined according to the situation of the cells;

2) Using high-purity DNA helps to obtain higher transfection efficiency;

3) DNA and transfection reagents are required to be diluted with the serum-free medium when preparing transfection complexes;

4) Antibiotics cannot be added to the medium during transfection;

5) Reagents should be stored at 2-8°C, and care should be taken to avoid repeatedly opening the lid for a long time;

6) The DNA concentration and the number of cationic liposome reagents should be optimized for the first use to obtain the maximum transfection efficiency. The ratio of DNA to transfection reagent is generally recommended to be 1:2-1:3.

1.3 Q: Does it need to be terminated after transfection?

A: No need. Liposome complexes are stable for 6 hours. If the cell medium is not changed before transfection, in order to ensure the nutrients required for normal cell growth, it is necessary to change to a new medium after 4-6 hours. However, if the medium has been changed before transfection, it is not necessary to change the medium after liposome transfection.

1.4 Q: Can co-transfection of DNA and siRNA be performed? How's the effect?

A: Yes, when DNA and siRNA are co-transfected, the siRNA transfection efficiency will be slightly worse.

1.5 Q: Can the transfection reagent be used for the transfection of lentiviral packaging?

A: Lentiviral packaging is possible.

1.6 Q: Can suspension cells be transfected with Hieff Trans™ Liposomal Nucleic Acid Transfection Reagent?

A: Hieff Trans™ Liposome Nucleic Acid Transfection Reagent can be used for suspension cell transfection, see Protocol for details. In addition, we also introduced a transfection reagent specifically for suspension cells (Cat No. 40805, Hieff Trans™ Suspension Cell-Free Liposomal Transfection Reagent)

2 Hieff Trans™ in vitro siRNA/miRNA Transfection Reagent

2.1 Q: Does the transfection reagent need to be changed after transfection?

A: This problem can be divided into two cases: 1. If there is no medium change before transfection, the medium should be changed about 6 hours after transfection to ensure the nutrients required for cell growth; 2. If there is a medium change before transfection , can be operated according to the normal operation of cultured cells? ? After the liquid change operation?

2.2 Q: Can transfection reagents be frozen?

A: It cannot be frozen, because the transfection reagent is a PEI cationic transfection reagent. Freezing at low temperatures will destroy the activity of the PEI transfection reagent. Therefore, it is best to store it at 2-8 °C to maintain the best transfection. efficacy.

Product information

Product name	SKU	Specifications
Hieff Trans™ Liposomal Transfection Reagent	40802ES02	0.5 mL
	40802ES03	1.0 mL
	40802ES08	5×1mL
Hieff Trans™ Suspension Cell-Free Liposomal Transfection Reagent (Inquire)	40805ES02	0.5 mL
	40805ES03	1.0 mL
	40805ES08	5×1 mL
Hieff Trans™ in vitro siRNA/miRNA Transfection Reagent (Inquire)	40806ES02	0.5 mL
	40806ES03	1.0 mL
Polyethylenimine Linear(PEI) MW40000（rapid lysis）	40816ES02	100 mg
	40816ES03	1 g
	40816ES08	5×1 g

Some of the articles published using our products

[1] Liu R, Yang J, et al. Optogenetic control of RNA function and metabolism using engineered light-switchable RNA-binding proteins. Nat Biotechnol. 2022 Jan 3. (IF:55)

[2] Luo J, Yang Q, et al. TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile. Cell. 2022 Mar 17.（IF:41.582）

[3] Zhou J, Chen P, et al. Cas12a variants designed for lower genome-wide off-target effect through stringent PAM recognition. Mol Ther. 2022 Jan 5.(IF:11.454)

[4] Chen S, Cao X, et al. circVAMP3 Drives CAPRIN1 Phase Separation and Inhibits Hepatocellular Carcinoma by Suppressing c-Myc Translation. Adv Sci (Weinh). 2022 Jan 24.(IF:16.808)

[5] Gu C, Wang Y, et al. AHSA1 is a promising therapeutic target for cellular proliferation and proteasome inhibitor resistance in multiple myeloma. J Exp Clin Cancer Res. 2022 Jan 6.(IF:11.161)

[6] Zhang Y, Yu X, et al. Splicing factor arginine/serine-rich 8 promotes multiple myeloma malignancy and bone lesion through alternative splicing of CACYBP and exosome-based cellular communication. Clin Transl Med. 2022 Feb.(IF:11.492)

[7] Qin J, Cai Y, et al. Molecular mechanism of agonism and inverse agonism in ghrelin receptor. Nat Commun. 2022 Jan 13.(IF:14.9)

[8] Tang X, Deng Z, et al. A novel protein encoded by circHNRNPU promotes multiple myeloma progression by regulating the bone marrow microenvironment and alternative splicing. J Exp Clin Cancer Res. 2022 Mar 8.(IF:11.161)

[9] Xie F, Su P, et al. Engineering Extracellular Vesicles Enriched with Palmitoylated ACE2 as COVID-19 Therapy. Adv Mater. 2021 Oct 19. (IF:30.849)

[10] Liang Y, Lu Q, et al. Reactivation of tumour suppressor in breast cancer by enhancer switching through NamiRNA network. Nucleic Acids Res. 2021 Sep 7.(IF:16.9)

[11] Fan Y, Wang J, et al. CircNR3C2 promotes HRD1-mediated tumor-suppressive effect via sponging miR-513a-3p in triple-negative breast cancer. Mol Cancer. 2021 Feb 2.(IF:27.403)

[12] Dai L, Dai Y, et al. Structural insight into BRCA1-BARD1 complex recruitment to damaged chromatin. Mol Cell. 2021 Jul 1.(IF:17.97)

[13] Zhang K, Wang A, et al. UBQLN2-HSP70 axis reduces poly-Gly-Ala aggregates and alleviates behavioral defects in the C9ORF72 animal model. Neuron. 2021 Jun 16.(IF:17.17)

[14] Li T, Chen X, et al. A synthetic BRET-based optogenetic device for pulsatile transgene expression enabling glucose homeostasis in mice. Nat Commun. 2021 Jan 27.(IF:14.92)

[15] Yan F, Huang C, et al. Threonine ADP-Ribosylation of Ubiquitin by a Bacterial Effector Family Blocks Host Ubiquitination. Mol Cell. 2020 May 21.(IF:17.97)

[16] Sun X, Peng X, et al. ADNP promotes neural differentiation by modulating Wnt/β-catenin signaling. Nat Commun. 2020 Jun 12.(IF:14.911)

[17] Yang X, Wang H, et al. Rewiring ERBB3 and ERK signaling confers resistance to FGFR1 inhibition in gastrointestinal cancer harbored an ERBB3-E928G mutation. Protein Cell. 2020 Dec.(IF:14.872)

[18] Zou Y, Wang A, et al. Analysis of redox landscapes and dynamics in living cells and in vivo using genetically encoded fluorescent sensors. Nat Protoc. 2018 Oct.(IF:13.490)

[19] Hao H, Hu S, et al. Loss of Endothelial CXCR7 Impairs Vascular Homeostasis and Cardiac Remodeling After Myocardial Infarction: Implications for Cardiovascular Drug Discovery. Circulation. 2017 Mar 28.(IF:29.69)

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Transfection Reagent – Hieff Trans™ and PEI

Linear PEI MW 40000, a more efficient transfection reagent