Figure 2. Effective Clean-Up to Produce High Quality Total RNA Compatible to Bioanalyzer Analysis.   Total RNA was isolated from 0.75 million HeLa cells using a competitor's phenol-based RNA extraction reagent. The resulting RNA was then purified using Norgen's RNA Clean-Up and Concentration Kit. As controls, total RNA was extracted using both Norgen's Total RNA Purification Kit (#17200, no phenol required) or the phenol-based RNA extraction reagent only without clean-up. Resolution of 1 µL of the 50 µL purified RNA on an Agilent RNA Nano 6000 Chip showed that without clean-up, the RNA sample isolated using the phenol-based RNA extraction reagent only resolved poorly on a bioanalyzer. In contrast, RNA extracted with the phenol-based RNA extraction reagent followed by clean-up by Norgen's RNA Clean-Up and Concentration Kit resolved properly on the bioanalyzer showing excellent quality.

Figure 3.  Effective Concentration and Detection of Low Amounts of RNA Input. Norgen's RNA Clean-Up and Concentration Kit can effectively concentration RNA inputs in the picogram range. Increasing amounts of HeLa RNA in 50 µL input volumes were concentrated to 20 µL using Norgen's RNA Clean-Up and Concentration Kit. A 10 µL aliquot of the purified RNA was then used as the template in a qRT-PCR reaction to detect the S14 gene. The amounts indicated on the graph correspond to the amount of RNA that was used as the input for the qRT-PCR reaction, demonstrating the consistent performance of the kit even in the picogram range.

Figure 4. Effective Clean-Up to Produce High Quality RNA Transcripts. Norgen's RNA Clean-Up and Concentration Kit effectively cleans up RNA transcripts with high recovery. Three RNA transcripts (1-3) of different sizes (266n, 161n and 419n, respectively) were produced using Norgen's  RNA Clean-Up and Concentration Kit. The resulting RNA transcripts were then purified using Norgen's  RNA Clean-Up and Concentration Kit. Resolution of 7.5 µL of the 50 µL cleaned RNA (C) as well as an equal volume of the uncleaned input (I) on a 1X MOPS, 1.5% formaldehyde-agarose gel showed the RNA transcript was successfully cleaned-up by Norgen's  RNA Clean-Up and Concentration Kit.

Figure 5. Effective Clean-Up to Produce High Quality RNA Transcripts Compatible to Bioanalyzer Analysis. Norgen's  RNA Clean-Up and Concentration Kit effectively cleans up RNA transcripts with high recovery. Three RNA transcripts (1-3) of different sizes (266n, 161n and 419n, respectively) were produced using Norgen's  RNA Clean-Up and Concentration Kit. Resolution of 1 µL of the 50 µL cleaned RNA transcripts on an Agilent RNA Nano 6000 Chip showed that RNA transcripts cleaned by Norgen's  RNA Clean-Up and Concentration Kit resolved properly on the bioanalyzer showing excellent quality.

Figure 6. Excellent Quality of Concentrated RNA. Total RNA isolated from HeLa cells (2 µg) was concentrated to 8 µL using the RNA Clean-Up and Concentration Micro-Elute Kit. The excellent quality is indicated by the electropherogram generated using the Agilent 2100 Bioanalyzer (RIN > 9). The concentrated RNA is a true 'total RNA' as can be observed by the presence of small RNA species.

Figure 7. High Recovery of Concentrated RNA. Total RNA isolated from HeLa cells (17.5 µL) was concentrated to 8 µL using the RNA Clean-Up and Concentration Micro-Elute Kit resulting in a 2-2.2 fold increase in RNA concentration, as was measured by Agilent 2100 Bioanalyzer quantification.

Figure 8. Concentration of Total RNA. The indicated amounts of HeLa RNA were concentrated using the RNA Clean-Up and Concentration Micro-Elute Kit. For each input amount, a 17.5 µL volume was processed and the RNA eluted in 8 µL, resulting in a 2.2 fold concentration. Three microliters of each eluate were used in 10 µL RT reactions with the oligo-dT primer, followed by real-time PCR using 3 µL of the resulting cDNA and primers specific for the human RPS15 gene. On average, amplifications of the concentrated RNA reached threshold 1 Ct value before the input RNA samples, which was expected based on the concentration factor. The lowest input amount used (240 pg) was only amplified when concentrated (most right sample in figure).

Figure 9. Concentration of miRNA. The indicated amounts of HeLa RNA were concentrated using the RNA Clean-Up and Concentration Micro-Elute Kit. Three microliters of each 8 µL eluate were used in 10 µL RT reactions with the miR-21-SLR primer, followed by real-time PCR using 3 µL of the resulting cDNA and the forward primer specific for miR-21.

Figure 10. Concentration of RNA prior to Next Generation Sequencing (NGS) applications. Total RNA was purified from 200 µL of plasma collected on EDTA blood tubes using Norgen's Total RNA Purification Kit (Cat. 17200) and eluted in 50 µL of elution solution. The same RNA was also concentrated two-fold using the Micro-Elute RNA Column by eluting in 25 µL of elution solution. Five microliters of both the RNA without additional concentration and the 2X concentrated RNA were used as inputs to generate RNA libraries (using the NEBNext® Small RNA Library Prep Set for Illumina® and following manufacturer’s instructions) for small RNA NGS on the MiSeq (Illumina) platform. A) The prepared small RNA libraries were visualized on a 6% TBE polyacrylamide gel, where the library prepared with 2X concentrated RNA contained more ligated/indexed miRNA cDNA (147-160 bp) products than the library prepared using the RNA without concentration. B) The cDNA was extracted from excised gel bands and interrogated using the Agilent 2100 Bioanalyzer (High Sensitivity DNA Assay). As would be expected based on input, the small RNA library prepared with the 2X concentrated RNA sample was approximately two times more concentrated than the library prepared with RNA without prior concentration (39.8 vs 21.2 nM, respectively).