Figure 1. Sequential Isolation of Total RNA, Genomic DNA and Proteins from 5 x 10⁵ HeLa Cells.  Panel A is a 1X MOPS 1% agarose gel showing the RNA that was isolated from 2 different samples of HeLa cells. Lane M is Norgen’s 1Kb RNA Ladder, and Lanes 1 and 2 contain 3 µL out of the 50 µL elutions. Panel B is a 1% agarose gel showing the gDNA isolated from the same 2 HeLa cell samples. Lane M is Norgen’s UltraRanger 1 kb DNA Ladder and Lanes 1 and 2 contain 10 µL of each of the 100 µL elutions. Panel C is a 12% SDS-PAGE gel that contains the proteins that were isolated from the 2 HeLa cell samples. Lane M is a protein ladder and Lanes 1 and 2 contain 10 µL of the 100 µL elution of proteins that had been column purified. The RNA, gDNA and proteins are all intact and of the highest integrity and quality.

Figure 2.  High Quality of RNA with Complete Size Diversity.  Norgen’s RNA/DNA/Protein Purification Kit isolates high quality total RNA without splitting the sample. RNA was isolated from 0.8 million HeLa cells using Norgen's RNA/DNA/Protein Purification Kit and a market-leading multiple-analytes isolation kit. One microliter of the 50 µL RNA fraction was resolved on an Agilent RNA Nano 6000 Chip. Both the gel diagram (Left) and the individual electropherogram (Right) showed that Norgen's RNA/DNA/Protein Purification Kit isolates a better quality of RNA, as indicated by electropherogram trace and the higher RIN value. In addition, Norgen’s proprietary resin allows the isolation of the complete size diversity of total RNA including the small RNA fraction containing microRNAs. Moreover, unlike the competitor’s kit, Norgen RNA/DNA/Protein Purification Kit isolates all three molecules from the same sample without splitting of the lysate.

Figure 3. High Quality RNA and DNA.  RNA, DNA and proteins were isolated from 0.5 million HeLa cells using Norgen’s RNA/DNA/Protein Purification Kit. Three microliters of the isolated 50 µL RNA was used as the template in a 20 µL RT reaction with and without reverse transcriptase in the absence of DNase treatment. One microliter of the RT reaction was then used in a 20 µL PCR reaction to detect the GAPDH gene. There was no amplification in the absence of reverse transcriptase (Lanes 2 and 4), indicating that the RNA is free of genomic DNA. Lanes 1 and 3 contain the successful RT-PCR results when reverse transcriptase was used. One microliter of the 100 µL purified genomic DNA was also successfully used in a 20 µL PCR reaction using the same set of primers to detect the GAPDH gene (Lanes 5 and 6), indicating the high quality of the isolated DNA. The larger PCR product is obtained due to an extra intron in the genomic DNA. Lane M is Norgen’s CloneSizer 100bp DNA Ladder and Lane 7 is a control with no template. All PCR products (10 µL of each loaded) were resolved on a 1X TAE, 1.2% agarose DNA gel. Therefore RNA and DNA isolated using the kit are high quality and can be used in downstream applications.

Figure 4. Consistent Detection of Large mRNA, microRNA and Genomic DNA from the Same Sample.  Norgen’s RNA/DNA/Protein Purification Kit is the only kit available that concurrently isolates genomic DNA and all sizes of RNA including microRNAs without the use of phenol. RNA, DNA and proteins were isolated from 8 x 10⁵ HeLa cells using Norgen’s RNA/DNA/Protein Purification Kit in triplicate. One microgram of RNA was used in a 20 uL reverse transcription with oligo dT primer and miR-21 stem-loop primer. Three microliters of the RT reaction was used in 20 µL qPCR reactions for human S15 (for Large RNA) and miR-21 (for microRNA) genes, respectively. Also, 100 ng of gDNA was used in a 20 µL qPCR reaction for the 5S rRNA gene. All three genes studied showed efficient amplification within the expected range of Ct values. Moreover, as no phenol extraction and no splitting of samples was required, all replicates showed very similar Ct values suggesting that high consistency was maintained throughout the isolation of multiple biomolecules.

Figure 5. High Quality Total Proteins.  Norgen’s RNA/DNA/Protein Purification Kit provides an additional column purification step for effective concentration and clean-up of the isolated proteins. In contrast, most competing multiple analyte isolation products require inefficient protein precipitation or provide no purification procedure at all. Ten microliter of the 100 µL protein fraction of 0.8 million HeLa cells isolated by Norgen’s RNA/DNA/Protein Purification Kit and a competitor’s kit was resolved on a 12% SDS-PAGE protein gel (Left). Similarly the same protein fraction was tested by Western Immunoblotting probed with an anti-human GAPDH antibodies (Right). Proteins from both kits performed well in Western blot as shown by good intense signals. However, proteins isolated with Norgen’s kit showed better resolution on SDS-PAGE gel with sharper individual bands, suggesting a cleaner protein sample with less contaminants such as detergents or salts.