Quantification of miRNA by SYBR Green qPCR: Difference between revisions
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Added RNA tailing |
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[[ Category: Transcription ]] | [[ Category: Transcription ]] | ||
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see [[ SOP - Chloroform ]] for safety information about working with Chloroform. | |||
This is adapted from [http://dx.doi.org Yang ''et al'' 2010] | 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 ]] | ||
* 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''', dissolved to 50 uM | * Oligo dT Adapter Primer '''5'GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3''', dissolved to 50 uM | ||
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==Protocol== | ==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 | |||
* Reedissolved in 25 μL of water | |||
===Reverse Transcriptase=== | ===Reverse Transcriptase=== | ||
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** 1 uL of Oligo dT Primer | ** 1 uL of Oligo dT Primer | ||
** 1uL of dNTP | ** 1uL of dNTP | ||
** | ** 6uL of tailed RNA | ||
** Sterile water | ** 5 uL Sterile water | ||
* Heat at 65C for 5 minutes | * Heat at 65C for 5 minutes | ||
* Briefly centrifugre then add (per reaction): | * Briefly centrifugre then add (per reaction): | ||