Auxin regulated gene expression in Arabidopsis BICD123 Introduction Auxin was the first identified and arguably the most important plant hormone Indole 3 acetic acid the main auxin in plants regulates almost every aspect of plant growth and development including embryogenesis seedling growth vascular patterning and flower development Auxin exerts its biological functions largely by activating signal transduction pathways that ultimately regulate the expression of downstream target genes that control particular developmental processes The current model of auxin signal transduction suggests that binding of auxin to the F box protein Transport Inhibitor Response 1 TIR1 promotes the interaction between TIR1 and the negative regulators auxin indole 3 acetic acid proteins AUX IAA thereby facilitating the degradation of AUX IAA proteins through the ubiquitin related protein degradation machinery Degradation of AUX IAAs in response to an auxin signal is believed to allow auxin response factors ARFs to form ARF ARF homo or heterodimers that then bind to the cis auxin response elements to activate or repress gene expression Auxin responses can be monitored at molecular level by analyzing the expression of auxin inducible genes There are several ways to monitor auxin induced gene expression Here we analyze auxin inducible genes by microarray and by RT PCR Experiments We will analyze the mRNA levels of wild type Arabidopsis seedlings and auxin treated seedlings 1 RNA extraction Procedure 1 Grind about 100 mg tissue quickly and thoroughly with a blue pestle 2 Add 450 l Buffer RLT Vortex vigorously 3 Transfer the lysate to a QIAshredder spin column lilac placed in a 2 ml collection tube and centrifuge for 2 min at full speed Carefully transfer the supernatant of the flow through to a new microcentrifuge tube without disturbing the cell debris pellet in the collection tube Use only this supernatant in subsequent steps 4 Add 0 5 volume of ethanol 96 100 to the cleared lysate and mix immediately by pipetting Do not centrifuge Proceed immediately to the next step Note The volume of lysate may be less than 450 l due to loss during homogenization Note Precipitates may be visible after addition of ethanol This does not affect the procedure 5 Transfer the sample usually 650 l including any precipitate that may have formed to an RNeasy spin column pink placed in a 2 ml collection tube Close the lid gently and centrifuge for 15 s at 8000 x g 10 000 rpm Discard the flow through Reuse the collection tube in the next step If the sample volume exceeds 700 l centrifuge successive aliquots in the same RNeasy spin column Discard the flow through after each centrifugation 6 Add 700 l Buffer RW1 to the RNeasy spin column Close the lid gently and centrifuge for 15 s at 8000 x g 10 000 rpm to wash the spin column membrane Discard the flow through Reuse the collection tube in next step Note After centrifugation carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through Be sure to empty the collection tube completely 7 Add 500 l Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 15 s at 8000 x g 10 000 rpm to wash the spin column membrane Discard the flow through Reuse the collection tube in the next step 8 Add 500 l Buffer RPE to the RNeasy spin column Close the lid gently and centrifuge for 2 min at 8000 x g 10 000 rpm to wash the spin column membrane The long centrifugation dries the spin column membrane ensuring that no ethanol is carried over during RNA elution Residual ethanol may interfere with downstream reactions Note After centrifugation carefully remove the RNeasy spin column from the collection tube so that the column does not contact the flow through Otherwise carryover of ethanol will occur 9 Place the RNeasy spin column in a new 2 ml collection tube supplied and discard the old collection tube with the flow through Close the lid gently and centrifuge at full speed for 1 min Perform this step to eliminate any possible carryover of Buffer RPE or if residual flow through remains on the outside of the RNeasy spin column after step 8 11 Place the RNeasy spin column in a new 1 5 ml collection tube supplied Add 30 50 l RNase free water directly to the spin column membrane Close the lid gently and centrifuge for 1 min at 8000 x g 10 000 rpm to elute the RNA 2 Quantification of RNA Quantify RNA yield by spectrophotometric analysis using the convention that I OD at 260 nm equals 40 g RNA per mL The total RNA you isolated will be sent out for microarray analysis 3 First Strand cDNA synthesis Step I Primer bybridization Mix 11 L of total RNA 1 L oligo dT 100 pmol L Incubate the mixture at 70 C for 10 minutes Quick spin and put on ice for 5 minutes Step II Temperature adjustment Add 4 L first cDNA buffer 2 L 0 1M DTT and 1 L 10mM dNTP mix Mix the solution by pipetting Incubate the mixture at 42 C for 2 minutes Step III First strand synthesis Add 1 L SSII reverse transcriptase Mix well Incubate at 42 C for 1 hour 3 PCR Mix the following reagents 2 L 10x PCR buffer 2 L 2 5 mM dNTP 0 5 L 10 M primer I 0 5 L 10 L primer II 1 L Taq DNA polymerase 14 L water PCR Step I 94 C 2 minutes Step II 94 C 30 seconds Step III 58 C 30 seconds Step IV 72 C 45 seconds Back to Step II for 25 cycles Step V 72 C 1 minute 4 Agarose gel 1 5 agarose