Kiranmai Durvasula1, Jeff Roeder1, Travis Butts1
The extraction of sufficient amounts of of high-quality RNA is a crucial step in generating relevant downstream results. Among a variety of commercially available extraction kits on the market, we pit the performance of our Omega Bio-tek E.Z.N.A.® HP Total RNA Kit (R6812-01) against Qiagen's RNeasy® Plus Mini Kit (74134). Both the kits are capable of efficiently purifying up to 100 µg of total RNA (> 200 nt) from a single extraction along with effective column-based gDNA removal. RNA was isolated from tissue samples as well as cultured cells and the kit performance was evaluated based on 3 parameters -- quantity, quality and integrity. The results of the comparison study are reported here.
Total RNA was purified from either 12.5 mg of fresh mouse kidney or 2.2 x 106 human embryonic kidney cells (HEK293) using Omega Bio-tek's E.Z.N.A.® HP Total RNA Kit and Qiagen's RNeasy® Plus Mini Kit (74134). Experiments were performed on replicates of four each. The extraction workflow of both kits are depicted in Figure 1. The number of steps involved are comparable and the entire procedure took about 25 minutes with both kits.
The RNA yield was quantified using Thermo Scientific NanoDrop 2000c. DNA co-eluted with RNA was quantified using 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 QuantiFluour® dsDNA System to the total RNA estimated using Thermo Scientific NanoDrop 2000c.
RNA isolated was analyzed on Agilent TapeStation 2200, which measures the RNA integrity, displayed as RNA Integrity Number (RINe). RINe ranges from 0 to 10, with 10 indicating maximum RNA integrity. Based on recent studies, RINe 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.
Figure 1. RNA extraction workflow comparison.
Figure 2 shows the RNA yields obtained using Omega Bio-tek and Qiagen kits with tissue samples and cultured cell samples. RNA yields were 18.36 µg and 28.17 µg from the mouse kidney, and 17.32 µg and 19.96 µg from the HEK293 cells using the 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 was significantly better than that of Qiagen's (p < 0.01) for both of the sample types tested.
Table 1. Percent gDNA contamination of the isolated RNA.
Genomic DNA contamination in the eluate 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 the E.Z.N.A.® 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 RINe 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 RINe number was 10 irrespective of the kit used. Gel images along with RINe numbers and representative electropherograms for the sample types are shown in Figures 3 and 4. The individual RINe scores suggest that isolated RNA is of high quality and acceptable for various downstream applications.
|Figure 3. RNA integrity analysis - tissue sample.||Figure 4. RNA integrity analysis - cultured cells.
a) Representative electropherogram - Qiagen,
b) Representative electropherogram - Omega Bio-tek
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 of 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.
Figure 5. Price per preparation cost comparison.
|E.Z.N.A.® HP Total RNA Kit||R6812-00||5|
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