microRNA Purification Kit

Figure 2. Efficient Removal of Large RNA Species. Different RNA species were isolated from 10⁶ HeLa cells, resolved on an Agilent® Lab-On-A-Chip, and electropherograms were generated. Panel A contains all the RNA species present in 10⁶ HeLa cells as isolated with Norgen's Total RNA Purification Kit and acts as a control. Panel B and C contain RNA that was isolated from 10⁶ HeLa cells using Norgen’s microRNA Purification Kit. One microliter of the 50 µL purified RNA for each fraction was loaded. Panel B shows the large RNA species removed using the Large RNA Removal Columns, and no small RNA can be detected. Panel C shows the small RNA that is isolated using the microRNA Enrichment Columns and shows that there is no contamination of the small RNA with any large RNA species above 200 nt. This demonstrates the effective separation of the small RNA from the large RNA species using Norgen's microRNA Purification Kit.

Figure 3. Effective Small RNA Amplification by End-Point RT-PCR. Norgen's microRNA Purification Kit isolates small RNA that could be amplified in RT-PCR using the poly (A) polymerase extension method. Small RNA was isolated from 1 million HeLa cells, and 10 µL of the 20 µL isolated small RNA were polyadenylated in a 50 µL Poly-(A)-Polymerase reaction. Seven microliters of the polyadenylated RNA were used in a 20 µL reverse transcription reaction with a poly T adaptor primer. One microliter of the reverse transcription was used in a 20 µL PCR reaction with primers against the human microRNAs (miR-21) and 5S rRNA. Panel A shows the amplification of the miR-21 transcript from small RNAs while Panel B shows the 5S rRNA amplification from small RNAs. Lane 1 in both panels shows the results when total RNA isolated from 1 million HeLa cells using Norgen's Total RNA Purification Kit was used as a control. Lanes 2 and 3 contain the successful RT-PCR when the microRNA isolated using Norgen's microRNA Purification Kit was used as the template, and Lanes 4 contain the non-template control. The RT-PCR was successful for both reactions using the microRNA as the template. All PCR products were resolved on a 1X TAE, 3% agarose gel using Norgen's PCR Ranger as the molecular weight ladder.

Figure 4. Better Recovery of miRNAs by Norgen's microRNA Purification Kit. Norgen's microRNA Purification Kit recovers microRNA more effectively than its competitors. Small RNA was isolated from 0.75 million HeLa cells using Norgen's microRNA Purification Kit and a competitors' kits. Relative expression of (A) miR-21, (B) miR-19, and (C) 5S rRNA was determined by RT-qPCR of polyadenylated total RNA samples. RT-qPCR was performed according to Shi and Chiang (2005). Fifteen microliters of the 50 µL isolated RNA were polyadenylated in a 50 µL Poly-(A)-Polymerase reaction. Four microliters of the polyadenylated RNA were used in a 20 µL reverse transcription reaction with a poly T adaptor primer. One microliter of the reverse transcription was then used in a 20 µL qPCR reaction with primers against the human microRNAs (miR-19 and miR-21) and house-keeping small RNA (5S rRNA). The resulting threshold cycle (Ct) values were summarized in the graph. Blue = Norgen's microRNA Purification Kit; orange = Silica-based Competitor micro-RNA Kit; green = Silica-based Competitor Total RNA Kit; red = Phenol-based reagent protocol; yellow = No Template Control. Norgen's microRNA Purification Kit recovered more miRNAs (lower Ct) than the competitor's microRNA-specific Kit.