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RNA isolation is a critical step in molecular biology, allowing researchers to extract high-quality RNA for downstream applications like gene expression analysis. One widely used method is the SPINeasy RNA Kit from MP Biomedicals. This kit employs silica-membrane spin-column technology, enabling efficient purification of total RNA from various sources, including animal tissues, plant tissues, and tissue cultures. The inclusion of specially formulated Lysis Buffer R and Lysing Matrix A facilitates rapid tissue sample lysis within seconds when combined with FastPrep instruments. This method provides a streamlined workflow, enabling the simultaneous processing of multiple samples without the use of toxic substances such as phenol and chloroform. The resulting purified RNA is of high quality and integrity, making it suitable for applications like RT-PCR and other molecular analyses.
Materials Needed:
- SPINeasy® RNA Kit for Tissue by MP Biomedical
- Tissue samples
- RNase-free water
- Ethanol (provided with the kit or molecular biology grade)
- Disposable pipette tips
- Microcentrifuge tubes
- Centrifuge
- Vortex mixer
- RNase-free bench and pipetting equipment
Procedure:
- Sample Homogenization:
- Grind the tissue samples using a homogenizer or mortar and pestle.
- Add the tissue homogenate to a microcentrifuge tube.
- Lysis and RNA Binding:
- Add lysis buffer to the homogenized samples.
- Mix the samples thoroughly by vortexing.
- Incubate the samples at room temperature for a specified period.
- Add ethanol to the lysate to create optimal binding conditions for RNA.
- RNA Binding to the Column:
- Load the lysate onto the provided RNA binding column.
- Centrifuge the column, allowing the RNA to bind to the column matrix.
- Washing Steps:
- Wash the column with wash buffer to remove impurities and contaminants.
- Centrifuge the column after each wash to remove the wash buffer.
- RNA Elution:
- Elute the purified RNA from the column using RNase-free water or a provided elution buffer.
- Centrifuge the column after adding the elution buffer to collect the RNA.
Always follow the specific instructions provided in the SPINeasy® RNA Kit protocol for the most accurate and reliable results. If you have any doubts or encounter issues during the procedure, consult the kit manual or contact MP Biomedical technical support for assistance.
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