Leukocyte RNA Purification Kits
For research use only and NOT intended for in vitro diagnostics.
Leukocyte RNA Purification Kits
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Supporting Data
Figure 1. Consistent Isolation of High Quality Leukocyte RNA. Norgen's Leukocyte RNA Purification Kit isolates leukocyte RNA of high quality with great consistency. Total leukocyte RNA was isolated from 100 µL of hamster blood using Norgen's Leukocyte RNA Purification Kit. A total of 6 replicates were performed, and 7 µL of the 50 µL purified RNA was then resolved on a 1.2% formaldehyde-agarose gel. As can be seen, Norgen's kit not only isolated high and consistent yields of total RNA, but the RNA was also of high quality as evidenced by intactness of the major 28S and 18S rRNA.
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.
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Kit Specifications
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| Maximum Column Binding Capacity |
Up to 50 μg RNA
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| Maximum Loading Volume |
650 μL
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| Size of RNA Purified |
All sizes, including small RNA <200 nt
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| Minimum Blood Input |
10 μL
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| Maximum Blood Input |
2 mL or 3 x 106 leukocytes
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| Time to Complete 10 Purifications |
40 minutes
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| Input Type |
Blood from Human, Hamster, Mouse, Rabbit, and Rat
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| Average Yield Human Blood (500 μL) Hamster Blood (100 μL) |
1.5 μg 2.5 μg |
Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment. The RBC Lysis Buffer should be stored at 4°C upon arrival.
| Component | Cat. 21200 (50 preps) | Cat. 21250 (50 preps) | Cat. 37800 (2 x 96 preps) |
|---|---|---|---|
| RBC Lysis Buffer | 2 x 100 mL | 2 x 1 L | 2 x 90 mL |
| Buffer RL | 30 mL | 30 mL | - |
| Binding Solution | - | - | 2 x 40 mL |
| Wash Solution A | 38 mL | 38 mL | - |
| Wash Solution | - | - | 2 x 30 mL |
| Elution Solution A | 6 mL | 6 mL | - |
| Elution Solution | - | - | 2 x 20 mL |
| Enzyme Incubation Buffer | - | 6 mL | 1 |
| DNase I | - | 1 Vial | - |
| Mini Spin Columns | 50 | - | - |
| Lysate Homogenization Column | - | 50 | - |
| Single Cell RNA Column | - | 50 | - |
| RBC Lysis 96-Well Plate | - | - | 2 |
| 96-Well Filter Plate | - | - | 2 |
| Adhesive Tape | - | - | 4 |
| Collection Tubes | 50 | 100 | - |
| 96-Well Collection Plate | - | - | 2 |
| Elution Tubes (1.7 mL) | 50 | 50 | - |
| 96-Well Elution Plate | - | - | 2 |
| Product Insert | 1 | 1 | 1 |
Documentation
FAQs
Spin Column
- Incomplete lysis of leukocytes.
Ensure that the appropriate amount of Buffer RL was used to lyse the leukocyte pellet.
- Lysis of red blood cells was incomplete.
Ensure that the blood sample is collected with the appropriate amount of EDTA, which will prevent coagulation of the red blood cells and allow for proper lysis. Also check that the appropriate amount of RBC Lysis Buffer is added to the blood sample, and that it is mixed and incubated properly.
- Ethanol was not added to the lysate.
Ensure that 200 L of 96-100% ethanol is added to the lysate before binding to the column.
- Ethanol was not added to the Wash Solution A.
Ensure that 90 mL of 96 - 100% ethanol is added to the supplied Wash Solution A prior to use.
- An alternative elution solution was used.
It is recommended that the Elution Solution A supplied with this kit be used for maximum RNA recovery.
- The column has become clogged.
Do not exceed 2 mL of blood or 3 x 106
- Incomplete lysis of leukocytes.
Ensure that the appropriate amount of Buffer RL was used to lyse the leukocyte pellet.
- Lysis of red blood cells was incomplete.
Ensure that the blood sample is collected with the appropriate amount of EDTA, which will prevent coagulation of the red blood cells and allow for proper lysis. Improperly lysed red blood cells will clog the column.
- Amount of blood used exceeds kit specifications.
It is recommended that no more than 2 mL of blood or 3x 106 leukocytes be used in order to prevent possible clogging of the column.
- Centrifuge temperature is too low.
Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 15°C may cause precipitates to form that can cause the columns to clog.
- RNase contamination.
RNases may be introduced during the use of the kit. Ensure proper procedures are followed when working with RNA. Please refer to “Working with RNA” at the beginning of the protocol's user guide.
- Procedure not performed quickly enough.
In order to maintain the integrity of the RNA, it is important that the procedure be performed quickly. This is especially important for the Cell Lysate Preparation Step in the Animal Tissue protocol, since the RNA in animal tissues is not protected after harvesting until it is disrupted and homogenized.
- Improper storage of the purified RNA.
For short term storage RNA samples may be stored at -20°C for a few days. It is recommended that samples be stored at -70°C for longer term storage.
- Leukocyte pellets were too old.
Leukocyte pellets generated at the end of Step 1 may be stored for up to 2 weeks at -70°C and used in this procedure. It is not recommended that samples be frozen for longer than 2 weeks, as the integrity of the RNA may be compromised.
- RNA was not washed 3 times with the provided Wash Solution A.
Traces of salt from the binding step may remain in the sample if the well is not washed 3 times with Wash Solution A. Salt may interfere with downstream applications, and thus must be washed from the well.
- Ethanol carryover.
Ensure that the dry spin under Column Wash in the centrifugation protocol or the extended vacuum in the vacuum protocol is performed in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.
