Difference between revisions of "Quantification of miRNA by SYBR Green qPCR"
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Revision as of 20:56, 20 July 2015
This is adapted from Pfeffer et al 2015
Materials
- Purify total RNA, via the protocol: Purification of miRNA and mRNA with TRIzol
- Superscript III reverse transcriptase (Life Technologies Catalog # 18080093)
- Oligo dT Adapter Primer 5'GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3
- Reverse Adapter Sequence 5'GCGAGCACAGAATTAATACGACTCAC-3
- Mature miRNA Primer
Protocol
=Reverse Transcriptase
- Reverse-transcribed into first-strand cDNA using Superscript III transcriptase (Invitrogen) with the oligo-dT adapter primer
qPCR
- 40ng of cDNA was used as a template in each reaction.
- The reverse primer was from the adapter sequence: 5'GCGAGCACAGAATTAATACGACTCAC3' and the forward primers were specific to miRNA mature sequences.
- The SYBR Green-based real-time PCR was performed to quantify miRNA expression, and U6 can be used for normalization.
