Dojindo’s HilyMax transfection reagent, distributed by the Microtech company, was tested by Dr. Yuri D’Alessandra, a researcher at the Immunology and Functional Genomics laboratories of the Monzino Cardiology Center in Milan.
The HilyMax reagent test on HL-1 cells
The HilyMax transfection reagent test performed on immortalized tumor cell lines (HeLa), widely used in scientific research, and on atrial cardiomyocytes (HL-1) used for the study of many human heart diseases, has given significant and satisfactory results compared to the results obtained with other transfect agents. HeLa and HL-1 cells were seeded in 24-well plates, following the Dojindo protocol and transfected with 0.5 micrograms / well of eGFP pCDNA 3.1 vector (containing a human gene of interest).
Results beyond all expectations
Since a very weak green fluorescence signal was detected by fluorescence microscopy, the evaluation of the expression of the GFP and the mRNA of the gene of interest was made by RT-qPCR. Both HeLa and HL-1 showed a very strong expression of the gene of interest. It should be emphasized that, by transfecting a human ORF, it was possible to determine that 100% of the levels of gene expression detected were due to the success of the transfection. The experiment was performed a second time with HL-1 cells in triplicate with overlapping results.
No previous attempt to transfect HL-1 cells with transfectants or electroporation has yielded comparable results.
To extract RNA from biological samples, specific reagents and kits based on the use of columns (spin column) are used. No RNA isolation method, however, ensures that RNA is free of genomic DNA contamination (gDNA). The presence of gDNA in RNA samples represents a problem when RNA must then be used in applications such as RT-PCR and qRT-PCR. False positives and alterations in the expression of genes are the result of a use of RNA contaminated with gDNA. Hence the need to eliminate gDNA contamination from RNA samples. In vitro digestion with deoxyribonuclease I (DNase I) is the most effective method for removing gDNA contamination in RNA samples. Unfortunately, the removal or inactivation of this enzyme after digestion is problematic.
The most commonly used methods for inactivating the enzyme (thermal inactivation, chelating agents such as EDTA, treatment with proteinase K followed by extraction with phenol / chloroform) are ineffective and even harmful to the integrity of RNA, in addition the fact that large quantities of RNA are lost (think of when you have available small quantities of RNA obtained from small biological samples). An alternative is represented by the use of DNase directly on the spin column, although this method is not efficient for samples with large quantities of DNA, such as spleen and thymus for which in vitro treatment is required.
Abmgood’s AccuRT Genomic DNA Removal Kit is based on a very simple and quick method (10 min) and effectively eliminates gDNA without loss or degradation of RNA. Thermal inactivation of the enzyme is not required. The treated RNA is compatible with various applications such as RT-PCR, qRT-PCR, microarray, Northern and more.