Trizol protocol I am using, provides good total RNA concentration and quality but the miRNA spike (Cel-miR39) is not being amplified in qPCR.
Starting volume: 600microlitre Plasma
Elution: 40microlitre
Sample1 260/280 Ratio: 2.04 Yield: 81.6ng/ml
Sample2 260/280 Ratio: 2.03 Yield: 61.3ng/ml
Sample3 260/280 Ratio: 2.04 Yield: 129.4ng/ml
hi Shalini,
I used Trizole method according to SIGMA provided protocol to isolate total RNA from plasma it was useful and gave me good yield , another important note:
you should dilute RNA /miRNA samples and prepare several reactions to get the appropriate RNA/miRNA concentration per reaction.
hope these information will help you in your work.
Luma
Hi , I used trizole in my work on miRNA in leukemia patients , hope its helpful
Investigation of miRNA Makeup in Iraqi Patients with Chronic Lymphocytic Leukemia.
https://rjpbcs.com/pdf/2018_9(5)/[190].pdf
luma
Thank you for the answers, Luna.
By dilution do you mean that post extraction I should put various dilutions of RNA for cDNA conversion and then use these cDNAs for qPCR?? If yes, then do I dilute the RNA in Nuclease free water?
You total RNA concentration is pretty good. May be it's something wrong with your Spike miR control? At what step of RNA extraction did you add cel-miR-39 to your samples? Did you recently purchased this control miRNA or use some relatively old stock? Do you use additional controls?
We normally use Unispike 2-4-5 mixture for RNA extraction, Unispike 6 for RT reaction, and Unispike 3 as an inter-pate control.
Vasily A Yakovlev thank you for the help. The spike-in (cel-miR-39) is new but the primers are a bit old. I added the spike-in to the trizol before starting the extraction. I intend to use cel-miR-39 as a control for extraction and Unispike6 as a control for RT reaction. Unispike 2-4-5 is also available with me. Sir, is there anything that I can do to check if I have extracted enough miRNAs apart from Nanodrop?
If I use Qubit, what concentration I can expect?
Shalini Dubey, when we expect high level of large RNA in our samples we use Agilent 2100 bioanalyzer with Small RNA chips to measure the miRNA fraction level. However, it's quite expensive and not always work good enough. Normally we don't do it for plasma or exosomal RNA.
The easiest way is to check expression of 4-5 real miRNAs and see what Ct value you have for them. From my experience miR-1260a, miR-2110, miR-21-5p, miR-320a, miR-328-3p, miR-451a, miR-486-5p, and miR-92a-3p have good expression level in many different plasma samples. Try primers to some of them for your qPCR.
for recovery of miRNA and other short RNA, I'd advise mirVana or MagMax mirVana kit
Vasily A Yakovlev Sir I do have primers for miR-451a and miR-92a. Will perform a qpcr for these and let you know. Thank you again for all the guidance and help.
Alexander Sasha Vlassov Sir thank you for the suggestion but due to restricted budget I cannot procure a kit for this purpose. Is there any other method?
Shalini, I am not sure what protocol do you use. These are our modifications for the standard Trisol protocol:
1. Use 2.5-3.0 volumes of ethanol instead of 1.5.
2. Incubate mixture of ethanol and clear fraction at -80C for at least a few hours (you can do it over-night). Lower temperature, longer incubation = higher precipitation of your RNA;
3. Elute your RNA with warm water (+55C). Wait 3-5 min after you add water to columns and spin them down. You can repeat elution by adding eluate back to the column and run it again.
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