Dear all,
Do you know any reliable and fast method to measure the levels of plant small RNAs (21,22 and 24 mer)?
I am interested in only comparing the abundance of those small RNA between mock and treated plants.
I am intending to run polyacrylamide gel, then just use the EtBr or a safe chemical to visualize all RNA on the gel. but not sure if 10-15 ug total RNA would be enough to detect siRNA.
any suggestion would be much appreciated.. thanks in advance.
Thank you @Mattias R. For ur answer. Yes, Northern blot is probably the solution, but I am not aiming at measuring any particular si or miRna with specific probe. I just would like to see the abundance of small RNAs following treatment with a new VSR.
If you Don have a target to be measured. You can use a molecular weight cut off filters.. 330 dalton is for one base. Consider your small rna to be anything smaller than 40 bases or 30 bases. So a filter with 10kd cut off will elute all ur small RNA below 30 bases. Which u can quantify using qubit or nano drop. And also u may run a polyacrylamide gel with 15 % strength. Look for a band or bands below 10 kd
Hello.
First, you can reverse transcribe your miRNAs by some method like stem-loop strategy and then:
a) quantify relative abundance by qPCR. (Google:
Stem-Loop qRT-PCR for the Detection of Plant microRNAs).
b) if you have a chance for it, do a droplet digital PCR which gets you absolute quantification of your target molecules!
I hope these are helpful hints. Good luck!
Thank you all for your answers. My target is to measure the overall abundance of small RNA (total accumulation of. 21, 22 and 24 siRNA. I am not particularly interested in any specific miRNa. ThUS, I can’t us SPECIFC PRIMERS OR probe for this.
one way yo do this is to send sample for small RNA sequencing, which is costly and time consuming. The other was is to extract total small rna by some kind of kits, and run northern blot with the use of p32 to label the 5’ end of all small rna then just use a film on see how the accumulation is .
Agilent's Bioanalyzer Small RNA kit quantifies small RNA (0-200nt) and miRNA (0-40 nt). Would that be useful for comparing samples? But you'd need the Bioanalyzer.
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