I want to detect the the levels of precursor miRNAs. Although there are many studies that explain detection of pre-miRNAs in animals, I have not been able to find many examples of the same in plants.
Don't see why is should be that different in plants versus animals. The pre-mRNAs should be detectable with pre-mRNA specific primers. As long as you know the sequence of the pre-mRNA you should be able to design primers.
You could perform a test to figure this out, although I haven't tried this before. We use miScript from QIAGEN to convert RNA to cDNA. That kit, or others like it, use Poly A tails for the seeding to create the cDNA and then you use a Poly A tail universal primer as a reverse primer against one specific to your miRNA. You could use a series of primers walking along the genomic location of your miRNA to figure out where the pre-miRNA starts?
Hope that helps.
Using Real-time PCR is the best option to detecting the level of microRNA in plants.
# Stem-loop RT followed by end-point PCR: Stem-loop RT primers bind to the 3'
portion of miRNA molecules, initiating reverse transcription of the miRNA. Then, the RT product is amplified using a miRNA specific forward primer
and the universal reverse primer.
Steps:
1. Isolate small RNA from plant material (Tri reagent/miRNA isolation kit)
2. Mir-X cDNA synthesis: RNAs are poly(A)-tailed using poly(A)
polymerase, and then copied using a modified oligo(dT) primer and SMART MMLV Reverse Transcriptase.
3. miRNA quantification employing RT-qPCR: Mir-X miRNA qRT-PCR
SYBR Kits use a single-step, single-tube reaction to produce first-strand cDNA, which is then specifically and quantitatively amplified using a
miRNA-specific primer and SYBR Advantage qPCR chemistry.
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