Figure 2.  Higher Yield and Quality of Leukocyte RNA Isolated by Norgen's Leukocyte RNA Purification Kit.   Norgen's Leukocyte RNA Purification Kit isolates Leukocyte RNA that exceeds the yield and quality of competitors. Total RNA was isolated from 200 µL of hamster blood using Norgen's Leukocyte RNA Purification Kit and a leading competitor’s kit. One microliter of the 50 µL purified RNA was resolved on an Agilent RNA Nano 6000 chip. The gel diagram (Upper panel) and the electropherogram (Lower panel) showed better quality of RNA isolated by Norgen's kit. In particular, RNA isolated by Norgen's Leukocyte RNA Purification Kit did not have any evidence of the RNA degradation that was present in RNA isolated by the competitor’s kit (Red Circle). In addition, Norgen's kit isolated higher amounts of RNA (Blue Line) with the additional recovery of small RNAs including miRNA (Black Circle) which were not present in RNA isolated by the competitor's kit. The above observation was further confirmed in RT-qPCR reactions as illustrated in Figure 3.

Figure 3.  High Yield of a Diversity of RNA Species.   Norgen's Leukocyte RNA Purification Kit effectively recovers all sizes of RNA from large mRNA to small RNA including microRNAs. Total RNA was isolated from 200 µL of hamster blood sample using Norgen's Leukocyte RNA Purification Kit and a leading competitor’s kit as illustrated in Figure 2. Two hundred nanograms of the purified RNA (both 50 µL elution volumes) was then used as the template in a RT-qPCR for detecting the beta-Actin gene (Lower Panel) and for detecting miR-21 (Upper Panel). In both graphs the blue lines correspond to Norgen isolated-RNA and the red lines correspond to competitor-isolated RNA. As it can be seen, Norgen's kit isolated higher yields of microRNA, as indicated by the lower Ct values of the blue lines (Upper Panel). Also, Norgen's kit successfully isolated a similar amount of the large RNA compared to the competitor’s kit (Lower Panel) indicating the full diversity of RNA species isolated.

Figure 4. Norgen's Leukocyte RNA Purification Plus Kit (Cat No. 21250) was used to Isolate RNA from 2.5 mL of Whole Blood Collected from 24 donors, Across 3 Experiments. Donors were tested in duplicate. After the standard protocol failed to produce RNA with sufficient concentration, modifications were made to the procedure and 16 additional donors were tested. 2 batches of 8 additional donors were tested in duplicate with the modified protocol for 32 separate RNA isolations; 32/32 yielded RNA concentration to >100 ng/µL. A single pass of 30 µL of elution buffer was used.

(Tessa Wiehle, Stephanie Borel, Andrew Kincaid, John Hedges, Jon Kemppainen, Justin T Brown, Andrew G Hadd, and Gary J Latham. Modifications to RNA Isolation Protocols Meet Requirements for Modern CML Monitoring of BCR-ABL1 Transcript Levels. Asuragen Inc. 2018)

Figure 5. Consistent Yield of High Quality Leukocyte RNA with Complete Size Range without the Use of Phenol. Norgen's Leukocyte RNA Purification 96-Well Kit allows for the consistent isolation of high quality RNA, with complete size range from the very large RNA down to small RNA without the use of phenol. Total RNA was isolated from 200 µL aliquots of hamster blood using Norgen's Leukocyte RNA Purification 96-Well Kit in 12 replicates. Five microliters of the 75 µL isolated RNA was resolved on a 1.2% formaldehyde agarose gel (Panel A) and 1 µL of the 75 µL isolated RNA was resolved on an Agilent RNA Nano 6000 Chip in a 2100 Bioanalyzer (Panel B). All 12 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 6. Isolation of Both Large and Small RNA. Norgen's Leukocyte RNA Purification 96-Well Kit allows for consistent isolation of both large and small RNA. Leukocyte RNA was isolated from 200 µL of hamster blood using Norgen's Leukocyte 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 miR-21 (Panel A) and the beta-actin 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.