Difference between revisions of "Quantification of miRNA by SYBR Green qPCR"

Revision as of 21:16, 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, dissolved to 50 uM
• dNTP Mixture (10 mM of each)
• 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
• Add to a PCR tube (this is per reaction):
  • 1 uL of Oligo dT Primer
  • 1uL of dNTP
  • 500 ng of RNA
  • Sterile water up to 13 uL
• Heat at 65C for 5 minutes
• Briefly centrifugre then add (per reaction):
  • 4 uL 5X First Strand Buffer
  • 1 uL 0.1M DTT
  • 1 uL RNAseOUT
  • 1 uL of Superscript III RT
• Mix gently by pipetting and place in the PCR machine for the following program:
  • Incubate at 50C for 60 min
  • Inactivate by heating at 70C for 15 min

qPCR

• Try 50ng of cDNA was as a template in each reaction (1/10th of the cDNA mixture).
• 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.
• Can use a protocol similar to the QPCR for mRNA quantification