Hello everyone,
I am new in research. I am having problem in removing DNA contamination from RNA isolated through TRizol method. I am working on miRNA overexpression on Alfalfa. Since miRNA size is very small, it is recommended to use TRIZOL extraction. I extracted RNA in 30ul of total volume from Alfalfa leaves. Checked their quality with nanodrop. Then I treated them with TURBO DNAse 1 with 2ug/ul RNA concentration. I tried doubling volume of DNAse, more incubation period, etc but results are very inconsistent. I am stuck, so please help me with your suggestions.
Thank you
Removing DNA contamination during miRNA (microRNA) isolation is crucial to obtain pure miRNA samples for downstream applications such as quantitative PCR (qPCR) or next-generation sequencing (NGS). Here are some steps you can take to minimize or eliminate DNA contamination during miRNA isolation:
Use DNase Treatment:
Add DNase I (RNase-free) to your RNA sample after the initial miRNA extraction step. DNase I will digest any contaminating DNA.
Incubate the sample with DNase I according to the manufacturer's instructions, typically at 37°C for 15-30 minutes.
Ensure Proper DNase Inactivation:
After DNase treatment, inactivate the DNase by adding an appropriate inactivation reagent (e.g., EDTA or heat inactivation) and incubate at the recommended temperature and time.
Use RNase-free Materials:
Use RNase-free tubes, pipette tips, and other labware to minimize the risk of introducing RNases, which can degrade RNA.
Perform a Mock Reaction:
Include a negative control sample in your miRNA isolation process. This sample should be processed in parallel but without the addition of reverse transcriptase or any other enzymes used in downstream applications. This control will help identify any residual DNA contamination.
Monitor DNA Contamination:
Assess the presence of DNA contamination by running an aliquot of your miRNA samples on an agarose gel or using a DNA-specific quantitative PCR assay. This will help you confirm the effectiveness of the DNase treatment.
Optimize RNA Extraction Method:
Consider using a miRNA-specific RNA extraction kit or method, as these are designed to selectively isolate miRNA and minimize DNA contamination.
Quantify RNA Purity:
Measure the RNA concentration and purity using a spectrophotometer or a specialized microRNA quantification kit. Verify that the A260/A280 and A260/A230 ratios are within the acceptable range for pure RNA.
RNA Integrity Assessment:
Assess the integrity of your RNA samples using gel electrophoresis or a bioanalyzer. Intact RNA should show distinct bands corresponding to miRNA species.
Repeat DNase Treatment If Necessary:
If you still detect DNA contamination after the initial DNase treatment, you may need to repeat the treatment or use a more rigorous DNase digestion protocol.
Use RNA Isolation Columns:
RNA isolation kits that use columns with RNA-binding silica membranes can effectively remove DNA contamination by washing away DNA during the purification process.
By following these steps and maintaining strict laboratory practices, you can minimize or eliminate DNA contamination during miRNA isolation, ensuring the purity of your miRNA samples for further experiments
When using Trizol, genomic DNA migrates to the interphase, which is between the aqueous phase (clear liquid on the top) and the organic phase (pink liquid on the bottom). It looks like a small, white, stringy phase. However, if your sample contains small DNAs, like plasmids, these will migrate to the aqueous phase, so the only way to remove them is with DNase treatment.
The best way to reduce genomic DNA contamination in your RNA is to avoid pipetting any of the interphase when you remove the aqueous phase. You said your final concentration of RNA is 2ug/ul which is a lot of RNA (although you don't say the volume), so my advice is to take less of the aqueous phase. If you only take about half of the aqueous phase then it's very unlikely you will accidentally take any of the interphase, and you should still get quite a lot of RNA based on your previous preps.
Thank you so much everyone, so basically i am working on the effect of overexpression of microRNA on photosynthesis and other abiotic climatic change resilience. Matthias R. Schaefer , as far as I know once we extract the RNA we RT to cDNA and see their expression in qPCR so if we do not remove the genomic DNA that might act as a template instead of our interested cDNA. Correct me if I am wrong.
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