Cloning of small RNAs with 5’ phosphate and 3’ OH ends

ThefollowingprotocoldescribesaprocedureforthepurificationandcloningofmiRNAsandothersmallRNAsinthe20-30nucleotidesizerangefromplanttissue.Figure1givesanoverviewofthecloningprocedure.

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Purifying20-30Nucleotidelengthsfrom10µgofTotalRNA(1µg/µl).

Insteadofgelpurification,mirVANAisolationcanreplacethisstepfollowingmanufacturer’sinstructions.Wehaveusedfrom1.5to5µgofsmallRNA(from10to35µgtotalRNA).Note,wealsouseArABIdopsisfloraltissue,whichhasahighpercentageofsmallRNAs(comment1).

1. Preparethe15%8MUREAgel.Poura15%UREAgelintheBioradMiniProtean2,using0.75mmspacers.

   12.5mlmakestwo0.75mmgels

   1. 5.25gUrea
   2. 625µl10XTBE
   3. 4.7ml40%acrylamide(19:1acrylamide:bis-acrylamide)
   4. Addwaterto12.5mlanddissolveurea
   5. Add87.5µl10%APS
   6. 4.4µlTEMED
   7. Pourimmediately(comment2)

2. Pre-runthegelfor30minat200Vandthenwashthewellsusing0.5XTBE.
3. Mix10µl(10µg)oftotalRNAwith10µlof2xGelLoADIngBufferII(orequalvolume100%formamidewithloadingdye)ina200µlRNase-freemicrofugetube.Heatthesampleat65°Cfor5min,thensnapcoolonice.Centrifugetocollectvolumetobottomoftubeandloadthesampleintoonewell.
4. Load10µloftheBaulcombeoligoladderalreadyprepared(sizesmarked20,30,40,60and80)
5. Runthegelat150Vuntilthebromophenolbluereachesthebottomofthegel(about2hours).Staintheladderportionofthegelwith0.5XEtBrfor2-5min.
6. Cutoutthegelslicecorrespondingto20-30nt(visualizedwithUV)withacleanrazorbladeandtransfertoa0.5mlRNase-freemicrofugetubewhosebottomhasbeenpunctured3to4timesbya21gaugeneedle.
7. Placethistubeintoa2mlRNase-freeround-bottommicrofugetubeandspinthegelthroughtheholesintothe2mltubeatfullspeedfor2min(comment3).
8. Add500µlofsterile0.3MNaCltothetubeandelutetheRNAbyrotatingthetubeovernightat4°C.
9. TransfertheeluateandthegeldebrisintoaSpin-XCelluloseAcetatefilterandspinatfullspeedfor2minutes.
10. Washthegelbitsoncemorewith100µl0.3MNaClandspinforanother2min.Collectthe100µleluateinthesametube.
11. Addanequalvolume(~600µl)of100%Isopropanoland3µlofglycogen(weusedAmbionglycoblue)tothesampleandincubateat–80°Cfor20to30minutes.
12. Spindownat14Krpmfor25minutesina4°Cmicrocentrifuge.
13. Carefullyremovesupernatantandwashpelletwith750µlofroomtemperature75%EtOH.AllowtheRNApellettoairdrythendissolvetheRNAintotalof5.7µlofDEPC-treatedwater(weusedAmbion’snuclease-freewater).

5’AdaptorLigationandPurification

1. HeattheRNAfor30secondsat90°Candthensnapcoolonice.
2. Setupthe5’Adaptorligationreactionina1.5mlRNasefreesilconizedmicrofugetube:
   • PurifiedmiRNAfromstep1.125.7µl
   • 5’RNAadaptor(5µM)1.3µl
   • 10XRNAligationbuffer1µl
   • T4RNAligase(10U/µl)1µl
   • RNAguard™(40U/µl)1µl
3. Incubateat37°Cfor1hour.(Orat+20°Cfor6hoursthen4°CovernightinaPCRmachine).
4. Stopreactionbyaddingeither10µl2xGelLoadingBufferIIor10µl100%formamideand3µlloadingdye.Heattheligatedsample/loadingbuffermixtureat65°Cfor5minutespriortoloadinggel.
5. Prerunthe15%TBUgel(0.5XTBE0.75mmBioradMini-ProteanIIgel)for30minutesat200V.Seerecipeinstep1.WashthewellswithTBEbufferpriortoloading.
6. Loadtheligatedsamplesandthe40-60ntladder(10µlofpreparedladder)inwellswithatleast1spaceinbetweenladderandsamples.ItisadvisabletoruneachsamplewithitsownladderandcutthegelbeforestainingORruneachsampleonseparategelstoavoidcontaminationamongstsamples.
7. Rungelat150Vuntilthexylenecyanolisnearthebottomofthegel(about2hours:thexylenecyanolrunsatabout40ntonthisgel).StaintheladderportionofthegelOReachsamplewithitsownladderusing0.5XTBE/EtBr.
8. Cutoutthegelbandcorrespondingto40-60ntwithacleanrazorblade(afteraligningthestainedladderbacktothegelifladderwascutawayseparately)andtransferthegelslicetoa0.5mlRNase-freemicrofugetubewhosebottomhasbeenpunctured3to4timesbya21gaugeneedle.
9. Placethistubeintoa2mlRnase-freeround-bottommicrotubeandspinthegelthroughtheholeintothe2mltubeatfullspeedfor2min.
10. Add500µlofsterile0.3MNaCltothetubeandelutetheRNAbyrotatingthetubeovernightat4°C.
11. TransfertheeluateandthegeldebrisintoaSpin-XCelluloseAcetatefilterandspinatfullspeedfor2minutes.
12. Washthegelbitsoncemorewith100µl0.3MNaClandspinforanother2min.Collectthe100µleluateinthesametube.
13. Addanequalvolumeof100%Isopropanol(~600µl)and3µlofglycogentothesampleandincubateat–80°Cfor20-30minutes.
14. Spindownat14Krpmfor30minutesina4°Cmicrocentrifuge.
15. Carefullyremovesupernatantandwashpelletwith750µlofroomtemperature75%EtOH.AllowtheRNApellettoairdrythendissolvetheRNAintotalof6.4µlofDEPC-treatedwater(weusedAmbion’snuclease-freewater).

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