I can only extraction about 1.5ug RNA from 3*10^6 mice peritoneal macrophages, which is much less than normal cells. How to raise yield?
I collect peritoneal macrophages by directly adding 1mL Trizol Reagent to a well to extract RNA. Rohan Ramesh Pawar
If you are keeping the cells in suspension then I would recommend you transfer them to conical tubes and spin at higher speeds to settle the cells and no loss of cell happens with this. Discard the cell culture supernatant and then add 1 ml Trizol to the pellet. I experienced similar problems in the past.
But if you keeping them in adherence, then remove the culture media and add trizol but here titutrate the cells for 10 times or more and then vortex for 2 minutes.
Vortexing is effective in both the approaches.
Chunlei Yuan Hope this helps.
Increasing RNA yield from mice peritoneal macrophages during the extraction process involves optimizing various steps to enhance the efficiency of RNA isolation. Here's a detailed guide on how to achieve higher RNA yields:
1. Cell Culture Optimization:
- Cell Density: Ensure that you start with an adequate number of cells. A higher cell density can contribute to increased RNA yield.
- Cell Viability: Use healthy and viable cells. Check cell viability before harvesting to avoid the inclusion of dead cells, which can degrade RNA.
2. Harvesting Cells:
- Timely Harvesting: Harvest cells at the appropriate time in the growth curve. This ensures that you collect cells when they are actively producing RNA.
- Use Gentle Detachment Methods: Employ non-enzymatic or gentle enzymatic methods for cell detachment to minimize cell stress.
3. Cell Lysis:
- Optimize Lysis Buffer: Choose a lysis buffer that efficiently breaks down cell membranes and releases RNA. Commercially available RNA extraction kits often provide optimized lysis buffers.
- Incorporate Protease Inhibitors: Add protease inhibitors to the lysis buffer to prevent RNA degradation by RNases.
4. Homogenization:
- Gentle Mechanical Disruption: If mechanical homogenization is necessary, use gentle techniques to avoid shearing RNA molecules. Dounce homogenizers or mild mechanical disaggregation can be suitable.
5. RNA Stabilization:
- Work Quickly: Perform the RNA extraction process swiftly to minimize RNA degradation.
- Use RNA Stabilization Reagents: Consider using RNA stabilization reagents during cell harvesting to preserve RNA integrity.
6. RNA Extraction Kit Selection:
- Choose a Suitable Kit: Select a high-quality RNA extraction kit that is suitable for macrophages. Kits optimized for challenging samples often include specific lysis buffers and additional features for improved RNA yield.
7. DNase Treatment:
- Include DNase Treatment: Remove genomic DNA contamination by incorporating DNase treatment during RNA extraction. This step ensures that the isolated RNA is free from genomic DNA, which can interfere with downstream applications.
8. Column Washes:
- Optimize Washing Steps: Follow the RNA extraction kit instructions for washing steps. Adequate washing helps remove impurities and contaminants, contributing to higher RNA purity and yield.
9. Elution Volume:
- Optimize Elution Volume: Elute RNA in an appropriate volume of elution buffer. Using a smaller volume can concentrate the RNA, leading to higher concentrations.
10. Quantification and Quality Assessment:
- Use Sensitive Quantification Methods: Use sensitive methods such as spectrophotometry or fluorometry to accurately quantify RNA.
- Assess RNA Integrity: Check the integrity of RNA using techniques like agarose gel electrophoresis or capillary electrophoresis (e.g., Bioanalyzer).
11. Repeat Extractions if Necessary:
- Repeat Extraction: If RNA yield is still suboptimal, consider repeating the extraction process using fresh samples.
12. Temperature Control:
- Maintain Cold Temperatures: Keep reagents and samples on ice or at low temperatures to slow down enzymatic activities that may lead to RNA degradation.
13. Standardize Protocols:
- Maintain Consistency: Standardize your RNA extraction protocol and follow it consistently to minimize variability.
14. Troubleshooting:
- Troubleshoot Issues: If you encounter low yields, troubleshoot the RNA extraction process by identifying and addressing potential issues systematically.
15. Collaborate and Seek Advice:
- Consult Experts: If possible, collaborate with experienced researchers or consult experts in RNA extraction techniques for specific cell types.
By systematically optimizing each step of the RNA extraction process, you can enhance the yield and quality of RNA extracted from mice peritoneal macrophages. Always validate the extracted RNA through appropriate quantification and quality assessment methods before proceeding to downstream applications.
What protocol are you using for RNA extraction? How many mice are you extracting from? When I plan an experiment (I have 3 treatment groups), I use 3-6 mice from each genotype and plate the cells on a 6 well plate, allow them to attach for 1h and then I immediately replace the media with SF media add the treatment and collect 24h later. I also extract RNA with Trizol, following the protocol (attached). I add the Trizol directly to the plate (no scraping, I pipette back and forth multiple times until the solution is thin) then I rotate at RT for 5 minutes before doing the extraction with Choloform. But, 1.5ug should be plenty of RNA to conduct cDNA synthesis and do the downstream experiments? I use a kit for cDNA synthesis and it requires 1ug of RNA, then I dilute the final cDNA then have 120ul of cDNA and each reaction for RT-PCR requires 1ul of the diluted cDNA. I have enough RNA to do the the cDNA synthesis at least 2 maybe 3 times. How much RNA do you need?
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