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) (updated with some clarifications) |
||
| (4 intermediate revisions by the same user not shown) | |||
| Line 2: | Line 2: | ||
[[ Category: Molecular Biology ]] | [[ Category: Molecular Biology ]] | ||
[[ Category: Transcription ]] | [[ Category: Transcription ]] | ||
| + | __NOTOC__ | ||
| − | + | see [[ SOP - Chloroform ]] for safety information about working with Chloroform. | |
| + | This is adapted from [http://dx.doi.org/10.1158/0008-5472.CAN-10-2579 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 ]] | ||
| + | * PolyA polymerase (NEB cat# M0276S) | ||
| + | * Phenol/Choroform mixture (1:1 ratio) | ||
| + | * 3M Sodium acetate pH 5.2 | ||
| + | * Isopropanol | ||
* Superscript III reverse transcriptase (Life Technologies Catalog # 18080093) | * Superscript III reverse transcriptase (Life Technologies Catalog # 18080093) | ||
| − | * Oligo dT Adapter Primer '''5'GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3''' | + | * miRNA Oligo dT Adapter Primer '''5'GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3''', dissolved to 50 uM |
| + | * dNTP Mixture (10 mM of each). Note this is not the normal 1 mM dNTP stock in the lab. | ||
* Reverse Adapter Sequence '''5'GCGAGCACAGAATTAATACGACTCAC-3''' | * Reverse Adapter Sequence '''5'GCGAGCACAGAATTAATACGACTCAC-3''' | ||
| − | * Mature miRNA Primer | + | * Mature miRNA Primer, this is a gene specific primer corresponding to the mature miRNA sequence. |
==Protocol== | ==Protocol== | ||
| − | ===Reverse Transcriptase== | + | ===PolyA Tailing of RNA=== |
| + | * Incubate at 37°C for 1 hour. Components include | ||
| + | ** 1 ug RNA | ||
| + | ** 2 uL 10X polymerase reaction buffer | ||
| + | ** 2 uL ATP (10 mM, comes with polymerase) | ||
| + | ** 1 uL poly(A) polymerase | ||
| + | ** ddH2O to a final volume of 20 uL | ||
| + | * Add 160 uL Water, 20 uL of 3 M sodium acetate, pH 5.2 and 200 uL of phenol/chloroform to tailed RNA, mix by tapping to form an emulsion | ||
| + | * Centrifuge for 1 min on max to separate layers | ||
| + | * Transfer the upper (aqueous) layer to a clean tube. Add 200 uL chloroform and mix, spin and extract aqueous layer to a new tube twice to remove the excess phenol | ||
| + | * Add 140 uL of isopropanol, mix and incubate 5 minutes. Centrifuge 15 minutes on max. Aspirate supernatant being careful not to touch the pellet and air dry for 5-10 mins | ||
| + | * Redissolved in 6 μL of water | ||
| + | |||
| + | ===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 an eppendorf tube (this is per reaction): | ||
| + | ** 1 uL of miRNA Oligo dT Adapter Primer | ||
| + | ** 1uL of dNTP | ||
| + | ** 6uL of tailed RNA | ||
| + | ** 5 uL Sterile water | ||
| + | * Heat at 65C for 5 minutes in the heating block. | ||
| + | * Briefly centrifuge then transfer to a PCR tube and 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 (called '''SS III Reaction'''): | ||
| + | ** Incubate at 50C for 60 min | ||
| + | ** Inactivate by heating at 70C for 15 min | ||
| + | * Can store the tailed cDNA at -20 until qPCR | ||
| + | |||
===qPCR=== | ===qPCR=== | ||
| − | * | + | * Generally use 100 ng of cDNA was as a template in each reaction (1/10th of the cDNA mixture). |
| − | * The reverse primer was from the adapter sequence: 5' | + | * The reverse primer was from the adapter sequence: 5'-GCGAGCACAGAATTAATACGACTCAC-3' 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 | ||
Latest revision as of 15:24, 1 March 2017
see SOP - Chloroform for safety information about working with Chloroform.
This is adapted from Yang et al 2010
Materials
- Purify total RNA, via the protocol: Purification of miRNA and mRNA with TRIzol
- PolyA polymerase (NEB cat# M0276S)
- Phenol/Choroform mixture (1:1 ratio)
- 3M Sodium acetate pH 5.2
- Isopropanol
- Superscript III reverse transcriptase (Life Technologies Catalog # 18080093)
- miRNA Oligo dT Adapter Primer 5'GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3, dissolved to 50 uM
- dNTP Mixture (10 mM of each). Note this is not the normal 1 mM dNTP stock in the lab.
- Reverse Adapter Sequence 5'GCGAGCACAGAATTAATACGACTCAC-3
- Mature miRNA Primer, this is a gene specific primer corresponding to the mature miRNA sequence.
Protocol
PolyA Tailing of RNA
- Incubate at 37°C for 1 hour. Components include
- 1 ug RNA
- 2 uL 10X polymerase reaction buffer
- 2 uL ATP (10 mM, comes with polymerase)
- 1 uL poly(A) polymerase
- ddH2O to a final volume of 20 uL
- Add 160 uL Water, 20 uL of 3 M sodium acetate, pH 5.2 and 200 uL of phenol/chloroform to tailed RNA, mix by tapping to form an emulsion
- Centrifuge for 1 min on max to separate layers
- Transfer the upper (aqueous) layer to a clean tube. Add 200 uL chloroform and mix, spin and extract aqueous layer to a new tube twice to remove the excess phenol
- Add 140 uL of isopropanol, mix and incubate 5 minutes. Centrifuge 15 minutes on max. Aspirate supernatant being careful not to touch the pellet and air dry for 5-10 mins
- Redissolved in 6 μL of water
Reverse Transcriptase
- Reverse-transcribed into first-strand cDNA using Superscript III transcriptase (Invitrogen) with the oligo-dT adapter primer
- Add to an eppendorf tube (this is per reaction):
- 1 uL of miRNA Oligo dT Adapter Primer
- 1uL of dNTP
- 6uL of tailed RNA
- 5 uL Sterile water
- Heat at 65C for 5 minutes in the heating block.
- Briefly centrifuge then transfer to a PCR tube and 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 (called SS III Reaction):
- Incubate at 50C for 60 min
- Inactivate by heating at 70C for 15 min
- Can store the tailed cDNA at -20 until qPCR
qPCR
- Generally use 100 ng of cDNA was as a template in each reaction (1/10th of the cDNA mixture).
- The reverse primer was from the adapter sequence: 5'-GCGAGCACAGAATTAATACGACTCAC-3' 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
