I'm working on MiRNA research and I extracted miRNA by using a serum/plasma Qiagen kit specific for MiRNA extraction because my samples are plasma from Leukemia/Lymphoma patients. After that, I measured the yield by nanodrop and gave good results, but when I wanted to measure the integrity of miRNA there was no result, I tried several things, for example, new samples, control samples from a healthy person, making cDNA synthesis and the same thing there is no result I do not know where is the problem is from the extraction kit ? or from where ?
If you have any idea please tell me
There are a few potential reasons for that:
- The Qubit fluorometer is designed to quantify nucleic acids by binding to the DNA/RNA backbone. However, it cannot assess integrity or size of the nucleic acids. So it is not the ideal tool to check miRNA integrity.
- miRNAs are very small compared to larger RNAs like mRNA. The Qubit assay may not be sensitive enough to accurately quantify these tiny RNA molecules.
- There could have been issues with the miRNA extraction method that resulted in low yields or degraded RNA. Though the Nanodrop gave good readings, it cannot distinguish intact vs degraded RNA.
Some recommendations on assessing miRNA integrity:
- Use an electrophoresis method like agarose gel, Bioanalyzer or TapeStation. These can separate RNA by size and show discrete miRNA bands vs smears of degraded RNA.
- PCR amplification of cDNA with primers spanning miRNA sequence - this requires intact miRNA to amplify.
- Northern blotting with probes specific to your miRNA of interest.
- qPCR using miRNAs with different GC content - degradation affects GC rich miRNAs more.
- Spike in a synthetic miRNA control before extraction to monitor preservation of small RNAs.
Salah A. Alshehade can I start my work depending on what I had on nanodrop to start making cDNA synthesis and after that panel of miRNA because the all idea from the research is doing the miRNA panel and see if there are differences between healthy control samples and patients samples?
Lugien Al-khalaileh It's understandable to be concerned when you are unable to assess miRNA integrity after extraction. However, this is actually expected with plasma/serum miRNA preps. Unlike larger RNAs like mRNA, miRNAs are too small (usually 18-22 nucleotides) to visualize by standard gel electrophoresis or bioanalyzer. The nanodrop readout is the primary quality metric.
Since you are getting good nanodrop readings for your miRNA extractions from plasma, it is very likely the extraction kit is working properly. Plasma contains very low amounts of miRNAs compared to tissues or cells, so yields in the 1-10 ng/μL range are common.
As long as the A260/A280 ratio is ~2.0 and A260/A230 is above 1.8, the miRNA prep should be of adequate quality to proceed with cDNA synthesis and qPCR-based miRNA profiling.
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