Figure 2. Amplification of Both Large and Small RNAs in the Same Extraction. Norgen's Total RNA Purification Kit Dx isolates the complete size range of RNA, including small RNAs without the use of phenol. Total RNA was isolated from 0.75 million HeLa cells using Norgen's Total RNA Purification Kit Dx, various silica-based competitors and phenol-based TRI Reagent. RT-PCR 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 used in a 20 µL qPCR reaction with primers against the human microRNAs (miR-19 and miR-21) and large mRNA (S15). Only the use of Norgen's Total RNA Purification Kit Dx resulted in detection of both microRNAs and mRNA amplification products similar to Competitor 2's kit but without the use of phenol.

Figure 3. High Quality of RNA from a Diverse Range of Inputs. Norgen's Total RNA Purification Kit Dx allows RNA isolation from a wide range of species or tissue types. Total RNA was isolated from 5 x 10⁸ E. coli cells, 1 x 10⁶ HeLa cells, 100 µL rat blood and 10 mg hamster kidney using Norgen's Total RNA Purification Kit Dx. One microliter of the 50 µL isolated RNA was analyzed on the Agilent® 2100 BioAnalyzer using an RNA Nano 6000 chip. Note the integrity of RNA from all inputs with the presence of small RNA species. Norgen's Total RNA Purification Kit Dx consistently isolates high quality RNA from various inputs that score a RIN value between 8 and 10.

Figure 4. Great Isolation Sensitivity. Norgen's Total RNA Purification Kit Dx allows sensitive RNA extraction from as little as a single cell. Total RNA was extracted from a decreasing number of 293 HEK cells from 1 million cells down to a single cell. Five microliters of the 50 µL isolated RNA was then subjected to a 20 µL reverse transcription using oligo dT primer. Three microliters of the reverse transcription was used in a 20 µL PCR reaction with primers to detect the human beta-actin transcripts. PCR products of beta-actin were detected from as little as a single cell. M is the marker lane.

Figure 5. Linear and Sensitive Isolation of Both Large and Small RNA. Norgen’s Total RNA Purification Kit Dx allows consistent isolation of both large and small RNA from different input amounts. Total RNA was isolated from 10 to 100,000 HeLa cells using Norgen's Total RNA Purification Kit Dx (blue), a competitor’s silica-based kit (green) and a phenol-based RNA extraction method (red). Relative expression of miR-21 (Panel A), and S15 (Panel B) was determined by RT-qPCR of total RNA samples. In brief, one microliter of the 50 µL isolated RNA was then subjected to a 20 µL reverse transcription using miR-21 stem-loop reverse primer or oligo dT primer. Two microliters of the reverse transcription was used in a 20 µL real-time PCR reaction with primers to detect the human miR-21 (Panel A) and the S15transcripts (Panel B). The resulting threshold cycle (Ct) values were plotted against input cell number. RNA isolated using Norgen's Total RNA Purification Dx had the best linearity (higher R²) and sensitivity (lower Ct) for both large RNA (S15) and small RNA (miR-21).

Figure 6. Effective and Consistent Detection of miRNA from Plasma. Norgen's Total RNA Purification Kit Dx can effectively isolate miRNA from plasma. Total RNA was isolated from 50, 100 or 200 µL of rat plasma in triplicates using Norgen's Total RNA Purification Kit Dx (blue), a competitor’s silica-based kit (green) and a phenol-based RNA extraction method (red). Stem loop RT-qPCR using primers specific to miR-21 was performed. In brief, two microliters of the 50 µL isolated RNA was then subjected to a 20 µL reverse transcription using miR-21 stem-loop reverse primer or oligo dT primer. Three microliters of the reverse transcription was used in a 20 µL real-time PCR reaction with primers to detect the human miR-21. Norgen's Total RNA Purification Kit Dx is the only product that showed (1) consistent detection of miR-21 transcripts across all input volumes, and (2) Ct values correlated to input volume (decrease Ct with increase input).

Figure 7. Recovering Diverse miRNA Species from Plasma with Better Consistency. Norgen's Total RNA Purification Kit Dx isolates plasma RNA that performs more consistently in downstream applications such as microarrays. Total RNA including miRNA was isolated from 100 µL of mouse plasma in duplicate using Norgen's Total RNA Purification Kit Dx, Competitor 1's leading miRNA Kit or a phenol-based RNA extraction method. One hundred nanograms of extracted RNA from each kit was applied to an Illumina microRNA expression profiling kit. Scatter plots display better consistency (better clustering) of replicate signals from Norgen's samples. Genes with Pval < 0.01 for both replicates were in blue. The associated table suggested that Norgen's protocol recovered the same diversity of miRNA with better consistency (higher r² value).

Figure 8. Better Diversity of miRNA Detected from Plasma. Norgen's Total RNA Purification Kit Dx isolates miRNA from plasma with better diversity than a leading competitor. Total RNA including miRNA was isolated from 100 µL of plasma using Norgen's Total RNA Purification Kit Dx or 625 µL of plasma using Competitor A's leading miRNA Kit, and was applied to an NCode expression profiling kit. Microarray images suggested that Norgen's Total RNA Purification Kit Dx (left) isolates a better diversity of miRNA from smaller input amount of plasma than the competitor’s miRNA kit (right). Image courtesy of LC Sciences, Houston.

Figure 9. Better Diversity of miRNA Detected from HeLa Cells using Illumina Small RNA Next Gen Sequencing. Norgen's Total RNA Purification Kit Dx isolates miRNA from HeLa cells with better diversity than a leading competitor. Total RNA including miRNA was isolated from 1 million HeLa cells using Norgen's Total RNA Purification Kit Dx and Competitor Q's leading miRNA Kit, and was applied to Illumina Small RNA Next Gen Sequencing on a MiSeq sequencer. Panel A showed that Norgen’s Total RNA Purification Kit Dx recovers a higher number of miRNAs than the competitor. In particular, the higher diversity is achieved with a faster and simpler procedure in as little as 20 minutes of RNA sample preparation time without the use of phenol. Panel B is a scatter plot of the average RPM (reads per million) of the miRNAs detected used to compare Norgen and the competitor’s kit recovery of miRNA. Norgen’s Total RNA Purification Kit Dx recovered a significantly higher number of miRNAs that have higher RPM, as summarized in the graph insert as well as in Panel C.

Figure 10. Consistent Yield of High Quality RNA with Complete Size Range without the Use of Phenol. Norgen's Total RNA Purification 96-Well Kit allows for the consistent isolation of high quality RNA, with complete sizes ranging from very large RNA down to small RNA without the use of phenol. Total RNA was isolated from 0.5 mL aliquots of an overnight culture of E. coli (~5 x 10⁸ cells/mL) using Norgen's Total RNA Purification 96-Well Kit in 16 replicates. Five microliters of the 75 µL isolated RNA were resolved on a 1.2% formaldehyde agarose gel. All 16 replicates showed both high yield and high quality. In addition, all replicates showed effective recovery of all sizes of RNA including the small RNA (arrow).

Figure 11. High Quality of RNA from a Diverse Range of Inputs. Norgen's Total RNA Purification 96-Well Kit allows for RNA isolation from a wide range of species and tissue types. Total RNA was isolated from 8 mg of brain (Lanes A), kidney (Lanes B), liver (Lanes C), lung (Lanes D) and spleen tissue (Lanes E), 7.5 x 10⁵ HeLa (Lanes F) and CHO cells (Lanes G), and 5 x 10⁸ bacteria (Lanes H) using Norgen's Total RNA Purification 96-Well Kit. Five microliters of the 75 µL isolated RNA was resolved on a 1.2% formaldehyde agarose gel. From observing the formaldehyde-agarose gel, it can be seen that Norgen's kit can be used to successfully isolate total RNA, including small RNA species, from a broad range of sample types.

Figure 12. Isolation of Both Large and Small RNA. Norgen's Total RNA Purification 96-Well Kit allows for consistent isolation of both large and small RNA. Total RNA was isolated from samples of 5 x 10⁵ HeLa cells using Norgen's Total RNA Purification 96-Well Kit. Aliquots of each total RNA sample were then used in 2 different RT-qPCR reactions. Ten microliters of the 75 µL isolated RNA was subjected to a 20 µL reverse transcription using miR-21 stem-loop reverse primer or oligo dT. Three microliters of the reverse transcription was used in a 20 µL real-time PCR reaction with primers to detect the human miR-21 (Panel A) and the S15 transcripts (Panel B), respectively. Both the mRNA and microRNA were amplified in a consistent manner from all the samples, with low variability of the Ct values. Thus both types of RNA are being consistently isolated using this kit.

Figure 13. Great Isolation Sensitivity. Norgen's Total RNA Purification Kit allows for sensitive RNA extraction from even less than 10 cells. RT-qPCR was used to detect mRNA isolated from various input amounts of HeLa cells, from 100,000 down to a single cell, using Norgen's Total RNA Purification 96-Well Kit. Ten microliters of the 75 µL isolated RNA were then subjected to a 20 µL reverse transcription using oligo dT primers. Three microliters of the reverse transcription were used in a 20 µL real-time PCR reaction with primers to detect the human S15 transcripts. The resulting Ct values were plotted against the input cell number. Total RNA was isolated and detected linearly from as little as a single HeLa cell.