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

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(copied protocol from Pfeffer paper)
 
(Filled in details about reverse transcriptase)
Line 8: Line 8:
 
* 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 ]]
 
* Superscript III reverse transcriptase (Life Technologies Catalog # 18080093)
 
* Superscript III reverse transcriptase (Life Technologies Catalog # 18080093)
* Oligo dT Adapter Primer '''5'GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3'''
+
* Oligo dT Adapter Primer '''5'GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3''', dissolved to 50 uM
 +
* dNTP Mixture (10 mM of each)
 
* Reverse Adapter Sequence '''5'GCGAGCACAGAATTAATACGACTCAC-3'''
 
* Reverse Adapter Sequence '''5'GCGAGCACAGAATTAATACGACTCAC-3'''
 
* Mature miRNA Primer
 
* Mature miRNA Primer
Line 14: Line 15:
 
==Protocol==
 
==Protocol==
  
===Reverse Transcriptase==
+
===Reverse Transcriptase===
 
* Reverse-transcribed into first-strand cDNA using Superscript III transcriptase (Invitrogen) with the oligo-dT adapter primer
 
* 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===
 
===qPCR===
* 40ng of cDNA was used as a template in each reaction.  
+
* 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 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.
 
* 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

Revision as of 21:05, 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