I am looking to run some qRTs on some isolated miRNA. I've used a kit to isolate the small RNAs from the total RNA. Is there a need for me to get a cDNA synthesis specific for miRNAs? If so, why? How are the small RNAs different from the larger RNAs for RT?
Additionally, will I need sybergreen/taqman kits designed for miRNA detection as well?
Thanks!
Answer to Question 1: If you are opting for Taqman based method, then you need to get miRNA specific cDNA, for SYBR green based method miRNA specific cDNA is not required.
Answer 2: In case of difference between small RNAs and large RNAs in terms of RT, usually oligo dT primers and/or degenerate primers are used for reverse transcription of mRNA, oligo dT primers for the known reason of polyA tail in mRNA. For mature miRNA's its polyadenylated and then reverse transcribed with oligo dT primers with universal tag in case of SYBR green based methods.
In case of Taqman based method, specific looped RT primer is used in RT for generating miRNA specific cDNA.
Answer 3: Taking into consideration the cost, time and sample decide to use SYBR green based kit OR Taqman kit.
Answer to Question 1: If you are opting for Taqman based method, then you need to get miRNA specific cDNA, for SYBR green based method miRNA specific cDNA is not required.
Answer 2: In case of difference between small RNAs and large RNAs in terms of RT, usually oligo dT primers and/or degenerate primers are used for reverse transcription of mRNA, oligo dT primers for the known reason of polyA tail in mRNA. For mature miRNA's its polyadenylated and then reverse transcribed with oligo dT primers with universal tag in case of SYBR green based methods.
In case of Taqman based method, specific looped RT primer is used in RT for generating miRNA specific cDNA.
Answer 3: Taking into consideration the cost, time and sample decide to use SYBR green based kit OR Taqman kit.
for miRNA isolation mirVana and PureLink kits work well. For miRNA qRT-PCR there are special TaqMan assays. the mature miRNAs (~18-25 nt) are very short compared to eg mRNA, and thus standard approaches wont work
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