Leukocyte RNA Purification Kits
For the rapid extraction and purification of total RNA from leukocytes
For research use only and NOT intended for in vitro diagnostics.
Leukocyte RNA Purification Kits
For the rapid extraction and purification of total RNA from leukocytes
Overview
- Fractionate leukocytes from whole blood in minutes with provided RBC Lysis Buffer
- Isolate total RNA, including microRNA, without phenol
- Rapid and convenient spin-column format and 96-well plate for high throughput applications
- Purified RNA is ready for any downstream application including RT-PCR, qRT-PCR, NGS, arrays and more. Find out more information on Norgen's NGS services
- Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix
These kits provide a rapid method for the isolation and purification of total leukocyte (white blood cell) RNA from mammalian blood samples. These kits are supplied with an RBC (red blood cell) Lysis Buffer for selective removal of red blood cells and fractionation of leukocytes by centrifugation. Isolation of leukocyte RNA results in improved expression profiling and other downstream applications by removing the masking effects of some RNAs which are very abundant in whole blood, such as globin mRNAs. These kits are able to isolate total leukocyte RNA, including both large mRNA and all small RNA species containing microRNA (miRNA) and small silencing RNA (siRNA). The purified RNA is of the highest quality and can be used in a number of downstream applications.
Leukocyte RNA Purification Kit (Spin Column)
This kit provides a rapid method for the isolation and purification of total leukocyte RNA from mammalian blood samples in 40 minutes. Allowable blood input ranges from 10 μL to 2 mL or 3 x 106 Leukocytes
Leukocyte RNA Purification Plus Kit (Plus)
Norgen’s Leukocyte RNA Purification Plus Kit provides a rapid method for the isolation and purification of total leukocyte (white blood cell) RNA from up to 3 mL of mammalian blood samples. Complete 10 purifications in as little as 40 minutes.
Leukocyte RNA Purification 96-Well Kit (High Throughput)
Purification is based on 96-well column chromatography using Norgen’s proprietary resin as the separation matrix. Purification can be performed using either a vacuum manifold or centrifugation. Norgen's kit allows for the isolation of total leukocyte RNA, including all small RNA species. The purified RNA is of the highest quality and can be used in a number of downstream applications including real time PCR, reverse transcription PCR, northern blotting, RNase protection and primer extension, and expression array assays. Allowable blood input ranges from 10 μL to 1 mL or 1.5 x 106 Leukocytes.
Details
Supporting Data
Kit Specifications
|
|
Maximum Binding Capacity Per Well
|
50 μg
|
Maximum Loading Volume Per Well |
500 μL
|
Size of RNA Purified |
All sizes, including
small RNA < 200 nt |
Maximum Blood Input* |
1 mL or 1.5 x 106 Leukocytes
|
Standard Blood Input |
150 μL
|
Minimum Blood Input |
10 μL
|
Average Yield: 500 μL human blood |
1.5 μg |
Time to Complete 10 Purification |
40 minutes
|
*Additional RBC Lysis Solution is required for input Volumes >150 µL
Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. The RBC Lysis Buffer should be stored at 4°C upon arrival. These reagents should remain stable for at least 1 year in their unopened containers.
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.
Citations
Title | Effects of Omega-3 Supplementation on Components of the Endocannabinoid System and Metabolic and Inflammatory Responses in Adipose and Liver of Peripartum Dairy Cows |
Journal | Research Square. 2022. |
Authors | Gitit Kra, Jayasimha Rayalu Daddam, Uzi Moallem, Hadar Kamer, Radka Kočvarová, Alina Nemirovski, G. Andres Contreras, Joseph Tam and Maya Zachut |
Title | MicroRNA molecular profiling from matched tumor and bio-fluids in bladder cancer |
Journal | Molecular Cancer. 2015. |
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Title | Novel Noncompetitive IL-1 Receptor-Biased Ligand Prevents Infection- and Inflammation-Induced Preterm Birth |
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Journal | Molecules. 2014. |
Authors | Balcerczyk, Gajewska, Macierzyńska-Piotrowska, Pawelczyk, Bartosz, Szemraj. |
Title | Increased TNF α, IL-6 and ErbB2 mRNA expression in peripheral blood leukocytes from breast cancer patients. |
Journal | Med Oncol. 2014. |
Authors | Alokail MS, Al-Daghri NM, Mohammed AK, Vanhoutte P, Alenad A. |
Title | Increased Il-4 Mrna expression and poly-aromatic hydrocarbon concentrations from children with asthma. |
Journal | BMC Pediatrics. 2014. |
Authors | Al-Daghri NM, Abd-Alrahman S, Draz H, Alkharfy K, Mohammed AK, Clerici MS, Alokail MS. |
Title | Targeted Inhibition of Serotonin Type 7 (5-HT7) Receptor Function Modulates Immune Responses and Reduces the Severity of Intestinal Inflammation. |
Journal | Journal of Immunology. 2013. |
Authors | Kim JJ, Bridle BW, Ghia JE, Wang H, Syed SN, Manocha MM, Rengasamy P, Shajib MS, Wan Y, Hedlund PB, Khan WI. |
Title | Biodosimetry on small blood volume using gene expression assay. |
Journal | Health Physics, 98(2):179-85. 2010. |
Authors | Brengues M, Paap B, Bittner M, Amundson S, Seligmann B, Korn R, Lenigk R, Zenhausern F. |