Application Note
Introduction
Materials and Methods
Total RNA was purified from either 12.5 mg of mouse kidney or 2.2×106 human embryonic kidney cells (HEK293) using Omega Bio-tek’s HP Total RNA Kit and Qiagen’s RNeasy Plus Mini Kit. Experiments were performed on replicates of 4 each. The extraction workflow of the kits is depicted in Figure 1. The number of steps involved are comparable and the entire procedure took about 25 minutes with both of the kits.
Quantity & Quality Assessment
The RNA yield was quantified using Thermo Scientific’s NanoDrop™ 2000c. DNA co-eluted with RNA was quantified using QuantiFluor® dsDNA system. The QuantiFluor® dsDNA system selectively binds to double-stranded DNA (dsDNA) in solution. It shows minimal binding to single-stranded DNA (ssDNA) and RNA, allowing specific quantitation of dsDNA. The percent gDNA contamination was estimated as the ratio of DNA quantified using QuantiFluor® dsDNA system to the total RNA estimated using Thermo Scientific’s NanoDrop™ 2000c.
Integrity Assessment
RNA isolated was analyzed on Agilent’s TapeStation® 2200, which measures the RNA integrity, displayed as the RNA Integrity Number (RIN). RIN ranges from 0 to 10, with 10 indicating maximum RNA integrity. Lately, RIN is being considered a de facto standard for RNA integrity as it takes into account the entire electrophoretic trace, not just the ratio of 28S and 18S rRNAs.
Results
Figure 2 shows the RNA yields obtained using the Omega and Qiagen kits with tissue samples and cultured cell samples. RNA yields were 18.36 μg and 28.17 μg from the mouse kidney, 17.32 μg and 19.96 μg from the HEK293 cells using Qiagen and Omega Bio-tek kits respectively. One-way ANOVA followed by Tukey’s post-hoc analysis indicates that the RNA yield extracted using Omega Bio-tek’s kit significantly better than that of Qiagen’s (p < 0.01) for both the sample types tested.
Genomic DNA contamination in the isolated DNA is shown in Table 1. With the RNeasy Plus Kit, DNA co-eluted was found to be 4.52% of total RNA for both the sample types tested. And it was 4.79% for HP Total RNA Kit for the same. The findings suggest that the RNA isolated from both the kits was of high quality with appreciable gDNA removal.
The purified RNA was also analyzed on the Agilent TapeStation® 2200 to provide information on its integrity. The average RIN numbers were 7.325 and 7.025 for the tissue samples (mouse kidney) using Qiagen and Omega Bio-tek kits, respectively. For cultured cells, the average RIN number was 10 irrespective of the kit used. Gel images, along with RIN numbers and representative electropherograms for the sample types used, are shown in Figures 3 and 4. The individual RIN scores suggest that isolated RNA is of high quality and acceptable for various downstream applications.
Discussion
The RNA yields were significantly higher from the tissue sample as well as the cultured cells isolated using the Omega Bio-tek kit than with the Qiagen kit. The cost with the Qiagen kit is $6.64 per preparation whereas it is $3.90 with Omega Bio-tek. For an end user, that is a significant 41% savings with Omega Bio-tek for comparable or better results.
