Difference between revisions of "Quantification of miRNA by SYBR Green qPCR"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (copied protocol from Pfeffer paper) |
Davebridges (Talk | contribs) (Filled in details about reverse transcriptase) |
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* 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=== | ||
| − | * | + | * 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
