Robert Adolf Brinzer
You performing a snap freezing and then grinding in a mortar and pestle step?
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Henry Kolge
Crushing the plant material in liquid nitrogen as mentioned above in mortar pestle and further isolation via trizol can give high yield of RNA
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Noor Rajeeb
Plant Tissue Selection: Choose plant tissues or organs that are rich in RNA and represent the specific RNA population of interest. Young and actively growing tissues tend to have higher RNA content. Pre-harvest Considerations: Ensure that the plants are healthy and free from stress factors that can affect RNA integrity, such as pathogens, drought, or extreme temperatures. Harvest the tissues quickly and handle them with care to minimize RNA degradation. Grinding or Homogenization: Grind or homogenize the plant tissue using liquid nitrogen or a suitable buffer to disrupt the cell walls and release RNA. Mechanical disruption methods such as bead beating or mortar and pestle can be used. Maintain the samples at a low temperature throughout the process. Lysis Buffer: Choose an appropriate lysis buffer that contains a strong denaturing agent like guanidine thiocyanate or guanidine isothiocyanate. These agents help in deactivating RNases and preserving RNA integrity. DNase Treatment: Incorporate a DNase treatment step to remove any genomic DNA contamination. DNase enzymes can be added to the lysis buffer or used during the RNA purification process. RNA Extraction Method: Select a suitable RNA extraction method based on your specific requirements. Common methods include phenol-chloroform extraction, silica column-based purification, or magnetic bead-based extraction. Each method has its own protocol and efficiency, so choose one that is known for high RNA yields and purity. Precipitation and Washing: Ensure that RNA is properly precipitated and washed during the extraction process to remove impurities. Follow the protocol carefully, including the use of appropriate washing buffers and centrifugation conditions. Quality Control: Assess the quality and quantity of the isolated RNA using spectrophotometry (e.g., measuring absorbance at 260 nm) and/or fluorescence-based methods. Additionally, perform a gel electrophoresis or use a bioanalyzer to check for RNA integrity. Storage: Store the isolated RNA at -80°C or in a suitable RNase-free storage buffer to maintain its stability and prevent degradation.
To obtain a high yield for plant RNA isolation, here are some general guidelines and tips:
It's important to note that the specific RNA isolation protocol and reagents used can vary depending on the plant species, tissue type, and downstream applications. Following established protocols, using high-quality reagents, and optimizing the extraction steps based on your specific plant sample will contribute to obtaining a high RNA yield.
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