Our ultimate goal is to measure microRNA gene expression. We have tried several kits including Qiagen and all gave poor values for RNA concentration and 260/280. Any suggestions or ideas would be appreciated. Thanks!
Hi Samuel,
In serum or plasma extracellular RNA concentration can be very low. So you need big amount of sample to get a good RNA yield.
The miRNAs and siRNAs not so long so they aren't used to be the target of the RNAses compare to long mRNAs.
The main thing in RNA isolation, is to protect your RNA agains RNAses. So you need to store your plasma or serum samples at -70 °C or lower (preferably at -80 °C) immediatly after sample collection. Or use any commercial RNA protecting reagent (like RNAlater (Thermo Scientific) or eNAT (Copan) or good old Trizol (TRI Reagent or similar brand names) which contain guanidine thyocianate as protecting agent.
The Trizol or any similar acid-guanidin-isothyocianate-phenol based lysis buffer effectively inhibit RNases in your samples. Then use the classical phenol-chloroform-isopropanol purifing method.
With the former method you can isolate high yield total RNA (include miRNA and small RNAs also). The purity is really depends on the last ethanol evaportaion steps effectiveness.
Alternatively you can use the Trizol lysate directly in Direct-zol RNA spin column kit https://www.zymoresearch.com/media/amasty/amfile/attach/_R2060_R2061_R2062_R2063_Direct-zol_RNA_MicroPrep_ver._1.0.0.pdf). I have very good experiences with this kit in human chordoma tissue RNA isolation in the terms of RNA quality and quantity. I have used the Trizole lysate after chloroform phase-separation step in the above kit. To decrease the protein contamination in the further purification steps
GITC/acidic phenol lysis combined with silica-based column methods
(Pros: after phase separation step no need to use chemical hood, can be high throughput Cons: more expensive than GITC-based methods
GITC/acidic phenol methods
(Pros: the cheapest, intact total RNA (including rRNAs, miRNAs) can isolated with high yield, Cons: toxic organic solvents (phenol, chlorophorm) (need a chemical hood), very problematic to use in high throughput).
Bests,
Tamas
Maybe as RNA in the extracellular environment..as a result is more prone to the extracellular insults resulting to such quality
Samuel C. Okpechi
Generally RNA extracted from biological fluids(serum, plasma, urine, etc) have low quality and quantity. I have tried different protocols and kits(LS TRIzol, Qiagen, Norgen) and all of them gave low ct value for internal controls. Finally I tried a RNA clean up and concentration kit( RNA Clean-Up and Concentration Micro-Elute Kit (Cat. 61000) https://norgenbiotek.com/product/rna-clean-and-concentration-micro-elute-kit) and it did improve ct values of both internal control and our gene of interest. you can use both phenol/chloroform or column based RNA extraction methods and concentrate your RNA using any kit like this.
You may know that guanidinium isothiocyanate (GITC) has strong but temporary denaturing effect while Beta-mercaptoethanol (ß-ME) will irreversibly denature RNases by reducing disulfide bonds and destroying the native conformation required for enzyme functionality(https://www.qiagen.com/cn/resources/faq?id=eac3139e-6c6c-4172-b61f-18d61b6cdd1e&lang=en). So, you may use Beta-mercaptoethanol to make sure RNAses are inhibited irreversibly.
Beta-mercaptoethanol must be added to the first buffer used in the kit I mentioned above. It may be one of the reasons we get high quality RNAs.
Another reason for low quality RNA from serum is the presence of inhibitors in serum. This may help you: http://jcmr.um.ac.ir/index.php/biology/article/view/31710
Optimizing microRNA quantification in serum samples. Journal of Cell and Molecular Research, 6(2), 52-56. https://doi.org/10.22067/jcmr.v6i2.31710
Dear Samuel Okpenchi,
Most of the Free-Circulating Plasma/Serum RNA is short RNA fragments. The A260:280 ratio is normally between 1 – 1.6. This low A260:280 ratio will not affect any downstream application. The use of mercaptoethanol in lysis is highly recommended to isolate RNA for sensitive downstream applications.
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