Detecting rare cells in circulatory system or bone marrow aspirates is difficult since such cells are diluted among many normal blood and bone marrow cells. However, the mRNA Isolation Kit for Blood/Bone Marrow and a companion reagent simplify the study of such rare cells (e.g., disseminated tumor cells for studying minimal residual disease) in several ways:
- The mRNA Isolation Kit for Blood/Bone Marrow increases the chance of finding transcripts from rare cells:
- It reproducibly yields large quantities of mRNA (variance in yield is less than 10%).
- It can concentrate the isolated mRNA up to 250-fold (from 5 mL of whole blood to a single 20 μL sample, which may be directly amplified in a single RT-PCR).
- It does notrequire an initial cell-separation step, thus minimizing the potential for loss of single or rare target cells.
- The mRNA Isolation Kit for Blood/Bone Marrow is intended for use with a companion reagent, the RNA/DNA Stabilization Reagent for Blood/Bone Marrow (sold separately), which stabilizes and lyses biological samples. This reagent immediately inactivates nucleases, as well as stabilizes cell lysates in the field for transport back to the laboratory. It also simplifies the study of rare cells by:
- Immediately lysing the cells, thereby "suspending" the metabolic state of target cells and the level of expressed mRNA in them. This shields the cellular nucleic acids from changes that might occur when cells are removed from their natural environment.
- Protecting mRNA (and DNA) from nucleases immediately after sample collection.
The mRNA Isolation Kit for Blood/Bone Marrow allows preparation of high-quality mRNA from stabilized blood or bone marrow samples.
Intended for life science research, the mRNA Isolation Kit for Blood/Bone Marrow is used to isolate mRNA from blood or bone marrow lysates preserved with the RNA/DNA Stabilization Reagent for Blood/Bone Marrow. The resulting yield of 50 – 200 ng mRNA per ml peripheral blood (normal donor) can be used for a variety of applications:
- Northern blotting
- Northern ELISA
- Ribonuclease protection assays
- cDNA library preparation
The intermediate nucleic acid fraction also can be processed by analytical techniques such as PCR. The mRNA Isolation Kit for Blood/Bone Marrow is particularly useful for the detection of rare cells like disseminated tumor cells by RT-PCR.
Features and Benefits
Easily scale the mRNA isolation up or down, the mRNA isolation up or down.
Isolate nucleic acids without prior cell separation, ensuring that mRNAs from all cells (even very rare cells) are represented in the final mRNA isolate.
Increase the efficiency of RT-PCR assays by removing PCR inhibitors (rRNA, tRNA, hemoglobin) from the mRNA template.
Obtain high yields of purified, intact mRNA, suitable for direct use in many research applications.
Increase the reliability and reproducibility of mRNA purification.
Easily stabilize blood or bone marrow using the RNA/DNA Stabilization Reagent for Blood/ Bone Marrow. Store samples for 12 months at -15 to -25°C or for one day at +2 to +8°C.
- Magnetic Glass Particles (MGPs)
- MGP Wash Buffer
- MGP Elution Buffer
- Hybridization Buffer
- Oligo (dT)20 probe, biotin labeled
- Streptavidin Magnetic Particles (SMPs)
- SMP Washing Buffer
- Double-distilled Water
The reagent is function tested in a model system that simulates the presence of five melanoma cells in 5 mL of normal human blood. Specifically, MelJu cell mRNA (5 pg) is diluted in a lysate prepared from 5 mL of fresh normal human blood. The lysate containing the target mRNA is first stabilized with the RNA/DNA Stabilization Reagent for Blood/Bone Marrow. Then the mRNA is purified from the stabilized lysate with the mRNA Isolation Kit for Blood/ Bone Marrow. Tyrosinase mRNA recovered by the kit is detected by RT-PCR.
Note: 5 pg of MelJu cell mRNA is equivalent to about five melanoma cells.
- The kit relies on magnetic glass particles and streptavidin-coated magnetic particles to isolate high-quality mRNA from stabilized peripheral blood and bone marrow aspirates.
- Lyse red blood cells and stabilize total nucleic acids with RNA/DNA Stabilization Reagent for Blood/Bone Marrow (separate reagent, not contained in the kit).
- Bind total nucleic acids to Magnetic Glass Particles and collect by centrifugation.
- Wash the Magnetic Glass Particles four times and collect them with a Magnetic Particle Separator after each wash.
- Elute total nucleic acids from the Magnetic Glass Particles. (Eluate may be used for DNA analysis.)
- Immobilize mRNA with biotinylated oligo(dT) and Streptavidin Magnetic Particles.
- Collect Streptavidin Magnetic Particles with a Magnetic Particle Separator and wash three times (as in step 3).
- Elute mRNA from Streptavidin-coated Magnetic Particles.
Figure 1: Stability of mRNA after lysis of whole blood cells in RNA/DNA Stabilization Reagent for Blood/Bone Marrow (research samples). A total of 6 mL human heparinized blood was lysed and stabilized with the RNA/DNA Stabilization Reagent for Blood/Bone Marrow. The lysate was divided into three aliquots which were stored either at +2 to +8°C, +15 to +25°C or -15 to -25°C. After 4 days, duplicate samples were taken from each aliquot and mRNA was isolated. The isolated mRNA was separated electrophoretically, transferred to a membrane by northern blotting, and analyzed with a DIG-labeled antisense beta-actin RNA probe.
Lanes A : Sample stored at +2 to +8°C
Lanes B : Sample stored at +15 to +25°C
Lanes C : Sample stored at –15 to -25°C
Result: The same band was clearly visible in each sample. However, the sample stored at –15 to -25°C (lanes C) contained more beta-actin RNA than the sample stored at +2 to +8°C (lanes A), which in turn contained more than the sample stored at +15 to +25°C (lanes B).
Figure 2: Isolation of mRNA from bone marrow and blood (research samples). Human heparinized blood and bone marrow were lysed using the RNA/DNA Stabilization Reagent for Blood/Bone Marrow. The mRNA was isolated from each lysate with the mRNA Isolation Kit for Blood/Bone Marrow. mRNAs corresponding to 0.8 mL of bone marrow and 1 mL of blood were analyzed by northern blotting using a DIG-labeled anti-sense RNA beta-actin probe.
A: bone marrow
Result: Comparable results were obtained with both blood and bone marrow. The single sharp band indicates that the isolated RNA was not degraded.
For life science research only. Not for use in diagnostic procedures.