Introduction: Additional Information: Reagents required: Basic Flow:

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1 ast update: 0//0 by R enome nalysis ore US elen iller amily omprehensive ancer enter Standard perating rocedure Title: icror Relative xpression S o.: 00 Version: ate: Total ages.: uthors:. opren, R. atson Reviewed:. opren ntroduction: icrors are small (~ nucleotides) endogenous Rs, that play important regulatory roles in animals and plants by targeting mr transcripts for cleavage or translational repression. ecause of their small size, they require special primers for reverse transcription. This increases the size of the mir in order to fit primers and a Taqman probe required for gene expression analysis. dditional nformation: n the reverse transcription (RT) step, c is reverse transcribed from total R samples using specific mir primers. The mir RT differs from regular RT in that it requires primers specific to the mir of interest. ach sample must have an RT reaction using the specific mir RT primer. This requires s icror Reverse Transcription it, an set of RT primers, and a Taqman (T) expression assay from. Reagents required: (in the RT fridge) R at ng/ul concentration, provided by user. Taqan icror RT it ( & ), 00 or 000 rxns. Taqan icror Reverse Transcription primer for each mir. (art of kit that includes the accompanying ). asic low: ) Set up plate lay-outs (RT & Relative xpression, ig. & ) ) ake RT aster ix ) liquot primers to strip tubes ) ipette RT (ul/rxn) + rimers (ul/rxn) + R (ul/rxn) = ul total/rt rxn ) ycle RT-R. Set-up late ay-outs. o the lay-out for the -well T relative expression plate first and then set up the corresponding lay-out for the RT, remembering that consecutive rows on the plate will be every other row on the. Use a -well plate document to lay out the RT (see igure and ). t may be beneficial to run only eight RT samples per row, as this is the easiest pipetting to a -well plate with triplicates.. ake RT master mix (ul/rxn) Rase Zap the workspace before beginning with procedure! Turn on the thermocycler, allowing the lid to heat up. alculate number of aster ix reactions to make: # aster ix rxns = Total # RT rxns + - extra

2 xample: total RT rxns + extra = 0 aster ix rxns ultiply each reagent by this number of reactions. ast update: 0//0 by R Table. RT aster ix xample Reagent X 0X 00m dts 0.. Reverse Transcriptase.0 0 0X RT uffer. Rase nhibitor 0.. d0 (ase & Rase ree).. Total Volume ul. liquot primers to strip tubes. (ul/rxn) rrange strip tube to be pipetted across the wells of the well plate. alculate volume of primer to add to each tube: Vol. rimer = (# samples per row X ul primer/sample) +.ul extra xample: ( samples/row X ul primer/sample) +.ul extra =.ul. dd this amount of each primer to its respective tube of the strip.. ipette master mix (ul/rxn), primers (ul/rxn), R(ul/rxn). ipette ul of aster ix to every well (ppendorf Repeater w/ 0.ml or 0.ml tip) ipette ul of primers across wells (. atrix ultihannel. ill ul, ispense ul. an use one set of tips since there is only in the wells.) ipette ul of R down columns. (. atrix ultihannel. ill ul, ispense ul. iscard tips after each use!) *R must be at ng/ul. t should be provided in strip tubes making it easy to align along the top of the well plate and pipette down sample columns.. ycle the reverse transcription. over the tubes or plate with the appropriate caps (plates are covered with bubble caps). oad the tubes or plates into the appropriate cycler ( or 00). - 00: the program is called mi-rna-rt. (must turn on in advance to heat lid!) - T-: the program is called R-RT. Run as follows: 0 min º - 0 min º- min º- º. (Total ~ min) igures: igure. RT-R lay-out in -well plate for assays and samples. 0 mir-.

3 ast update: 0//0 by R igure. Relative xpression lay-out for -well plate for assays and samples. mir- mir dditional nformation: Table. RT-R Reaction Reagent Volume er ul rxn inal onc. or nput RT rimers (X) ul X R (ng/ul) ul 0. ng RT aster ix ul (eed to make, see Table ) Reagents required: c from RT reaction, in strip tubes or a plate..reagents for relative expression aster ix (x T buffer, m gl, m dts, mplitaq old U/ul, all in -0 freezer. for each mir. (art of kit that includes the accompanying RT primers). asic low: ) heck plate lay-out ) ake aster ix ) ipette (ul/rxn) + (ul/rxn) + (.ul/rxn) to -well deep well plate ) ipette + + (.ul/rxn) to well plate ) ipette c (.ul/rxn) to well plate ) over, Spin, & ycle. heck that plate lay-out has been done with mir RT.

4 ast update: 0//0 by R. ake aster ix alculate number of aster ix reactions to make: # rxns = (# rxns/row + )(total # rows) + (0 - extra). xample: ( rxns/row + )( rows total) + extra = rxns ultiply each reagent by this number of reactions: Table. Relative xpression aster ix xample Reagent X X X T uffer 0 m gl.. m dts 0.. mplitaq old 0.. (u/µ) d0. 0. Total Volume ul. ipette (ul/rxn) + (ul/rxn) + (.ul/rxn) to deep well plate alculate the number of reactions worth of reagents () to add to a deep well: = (# reactions per mir assay) + extra xample: reactions per mir + extra = = ultiply by the volume of each reagent: Table. Reagents added to deep well xample Reagent X = X aster ix.. dd this volume of each reagent to the first column of the -well deep well plate: igure. -well eep Well late 0 mir-. ut on a temporary sticky cover with plate scraper & vortex plate gently (no bubbles!) Spin down at 00g for 0s.. ipette + + (.ul/rxn) to well plate ipette mix from deep well plate across a well plate. - Use atrix ultihannel. ill, ispense. (enough for columns)

5 ast update: 0//0 by R - This will leave ~ 0.ul after final dispense, purge back into deep well column - an use a single set of tips because there is no c in the wells. - f you only need wells: ill, ispense.. igure. well plate layout for pipetting mir- mir ipette c (.ul/rxn) to well plate ipette.ul c into wells (. atrix ultihannel. ill.0, ispense.) - fter final dispense for each sample, discard remaining volume - Use. filtered tips & change tips for new samples. over, Spin, & ycle over plate (optical cover and adhere with plate scraper) Spin down (000rpm for min) Run on 00 (same as regular Taqman: -0 min; 0 cycles of: - sec, 0 - min) dditional nformation: Reagent aster ix Volume/ 0 ul rxn inal concentration/ rxn X T uffer X m gl.. m m dts m mplitaq old units (u/µ) d0. Total Volume ul ote from training w/. athew on //0: -Total volume of T reaction is 0.0 ul because of pipetting limitations when pipetting c, but should be treated like 0 ul for calculations (explain somewhere?)