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

From Bridges Lab Protocols
Jump to: navigation, search
m (removed table of contents)
(Added link to Pfeffer article)
Line 3: Line 3:
 
[[ Category: Transcription ]]
 
[[ Category: Transcription ]]
 
__NOTOC__
 
__NOTOC__
This is adapted from Pfeffer ''et al'' 2015
+
This is adapted from [http://dx.doi.org Yang ''et al'' 2010]
 
==Materials==
 
==Materials==
 
* Purify total RNA, via the protocol: [[ Purification of miRNA and mRNA with TRIzol ]]
 
* Purify total RNA, via the protocol: [[ Purification of miRNA and mRNA with TRIzol ]]

Revision as of 18:38, 10 January 2017


This is adapted from Yang et al 2010

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