Primer/probe (AOD or IDT) mix 4µL

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1 Genome Analysis Core UCSF Helen Diller Family Comprehensive Cancer Center Standard Operating Procedure Title: TaqMan Relative Expression SOP No.: GC 003 Version: 3 Date: Total Pages: 7 Authors: L. Edmondson, L. Smythe, K. Copren Reviewed: K. Copren Introduction: Relative expression compares expression levels of genes of interest (GOI) to the expression levels of one or more endogenous control genes. The expression level is measured as a Cycles at Threshold (CT) value -- the cycle in which the florescence level crosses a threshold value of fluorescence, which occurs during the exponential phase of amplification. This CT value is directly correlated to the amount of starting transcript and therefore can be used to calculate relative amounts of starting transcript. Additional Information: TaqMan chemistry uses two PCR primers (a forward and a reverse) and a probe, which anneals to the middle of the amplicon. On the 5 - end of the probe is a florescent reporter molecule, typically FAM. On the 3 - end is a quencher molecule, typically Black Hole Quencher 1 (BHQ1) or TAMRA. While both the reporter and quencher are attached to the probe, the quencher inhibits reporter fluorescence. During PCR extension, however, the reporter is cleaved from the probe and resleased from the quencher. This allows the real-time PCR instrument to detect fluorescence from the reporter molecule. BHQ1 quencher probes normally have a dark purple color, while TAMRA quencher probes are bright pink. For the analysis, it is important to know which quencher was used. Basic Flow of Project: 1. Prepare plate layout 2. Perform master mix + cdna + dh20 calculations 3. Perform primer/probe mix calculations and prepare strip tubes 4. Make master mix 5. Make master mix + cdna + dh2o samples 6. Make primer/probe mix 7. Pipette master mix + cdna + dh2o samples onto plate 8. Pipette primer/probe mixes onto plate Example Project: This protocol refers to an example project that uses 4 genes and 16 samples. See layout in fig. 1. Total contents of reaction: Reagent Master mix 12µL cdna + dh2o 4µL Primer/probe (AOD or IDT) mix 4µL Total 20µL Volume Part 1. Prepare plate layout using 364-well format. (See fig. 1). 1

2 Write gene names on lines A-P; one gene per row. If possible, skip a row between each gene. Draw samples, remembering to include triplicates. Vertically, each sample should span each set of genes (ex. Genes 1-4); horizontally, each sample should span 3 wells. Because the Matrix Multichannel Pipette will only pipette in every other row, highlight every other row with one color and the remaining rows with another color. (See fig. 1). Part 2. Prepare reagents and perform master mix + cdna + dh2o calculations Reagent Master mix 5X TaqMan Buffer 12µL 25 mm MgCl 2 25 mm dntps dh2o TaqGold (5u/µL) cdna + dh2o 4µL Total 16µL Volume 1. Remove master mix reagents, primers, and probes from -20 freezer (Freezer I) and allow to thaw. Reagents, primers, and probes may be thawed at RT, but should then be placed on ice. AmpliTaq Gold should always be on ice. 2. Calculate the volume of master mix + cdna + dh2o needed. a. Calculate total # of reactions per sample (Total # genes/sample * 3 replicates) + x extra Ex. (4 genes/sample * 3) + 2 = 14 rxns/sample See chart below to determine x, which allows for pipetting error b. Calculate volume of master mix needed per sample Total # rxns/sample * 12µl master mix Ex. 14 rxns * 12µl master mix = 168µl master mix/sample c. Calculate volume of cdna needed per sample (Total # rxns/sample) * (ng cdna/rxn) * (µl/[cdna]) Ex. (14 rxns/sample) * (5ng cdna/rxn) * (1µl/50ng cdna) = 1.4µl cdna/sample cdna input is typically 5ng/rxn, but may vary with each project. cdna concentration should be known before beginning project or provided by us d. Calculate volume of dh2o, which brings total volume to 16ul [Total # rxns/sample * (16µl total vol/rxn 12µl master mix/rxn)] µl cdna Simplified: (Total # rxns/sample * 4µl dh2o/rxn) µl cdna Ex. (14 rxns/sample * 4µl H2O/rxn) 1.4µlcDNA = 54.6µl dh20/sample 2

3 # of reactions (# genes x 3) # of reactions to add (x) or more +4 or more *Note: Never pipette less than 1µl cdna. Add more reactions if needed. 3. Calculate total volume of master mix needed. (Total # rxns/sample) * (# samples) + x extra Ex. (14 rxns/sample) * 16 samples + 6 = 230 rxns of master mix Remember, the total # rxns/sample should already include x extra to allow for pipetting error For the master mix, x extra is typically 6-8, but may need to be increased as the number of reactions increases. For a full plate, for example, it may be necessary to add as many as 20 extra reactions. 4. Generate a table indicating the master mix reagent volumes needed for one reaction and for the total # of reactions. Ex: Reagent Volume (µl) per rxn Volume (µl) x230 rxns 5X TaqMan Buffer mm MgCl mm dntps dh2o TaqGold (5u/µL) Total 12µL 2760 *Note: Total volume/# rxns should equal 12µL. Part 3. Perform primer/probe mix calculations and prepare primer/probe mix tubes. IDT primer/probe mix Reagent Initial concentration Final Concentration Vol. Per Reaction F/R primers x (standard 50µM) x (standard 500 nm) x Probe x (standard 20 µm) x (standard 200 nm) x dh2o 4-x Total 4 µl AOD mix Reagent Initial concentration Final Concentration Vol. Per Reaction AOD 20x 1x 1 dh2o 3 3

4 Total 4 µl 1. Determine # primer/probe mixes needed. Prepare one mix for each row on your plate. Ex. A project with 4 genes and 8 samples requires 4 tubes of primer/probe mix. A project with 4 genes and 16 samples requires 8 tubes of primer/probe mix. (See fig. 1). Use one set of colored strip tubes for each highlighter color used. Label each tube with the corresponding gene name. Ex. Use one set of 4 orange tubes labeled 1, 3, 1, 3. Use one set of 4 yellow tubes labeled 2, 4, 2, Determine # rxns needed for each primer/probe mix. [(# samples tested with that primer mix) * 3 triplicates)] + 2 extra Ex. For a project with 4 genes and 8 samples, each primer/probe mix will contain (26) reactions. For a project with 4 genes and 16 samples, each primer/probe mix will still contain 26 reactions. However, make 2 sets of primer/probe mix. (See fig. 1). 3. For each primer/probe mix, calculate volume of F/R primer and volume of probe required to obtain desired final concentration. For IDT primer/probe sets: Primer: (Aliquot F/R conc.)(x µl input) = (final conc.)(20µl total vol) Ex. (50uM)(x µl input) = (500nM)(20µl total vol) x = 0.2µl of 50uM F/R primer Probe: (Aliquot probe conc.) (x µl input) = (final conc.)(20µl total vol) Ex. (20uM)(x µl input) = (200nM)(20µl total vol) x = 0.2µl of 50uM F/R primer Note: The standard aliquot concentration of F/R primer is 50uM. The standard final primer concentration is 500 nm (.5µM). The standard aliquot concentration of probe is 20uM. The standard final probe concentration is 200 nm (0.2µM). These values can vary with each primer and probe, however, and should be determined before beginning the project. Desired final primer and probe concentrations can be found on the Expression_Assays_Core_List_DG on the Sorted Expression Assays page. Note: The primer/probe mix will be 4ul, but concentrations should be calculated using the total volume of 20µl (16µl (master mix + cdna + dh2o) + 4µl primer mix). For an AOD: Always use 1µl of AOD and 3µl dh2o, as the AOD is 20x and the rxn. vol. is 20µl. 4

5 4. Calculate volume of water needed in primer/probe mix. 4µl total volume xµl F/R primer Ex. 4µl (0.2µl F/R primer µl probe) = 3.6µl dh2o 5. Generate a table of values, then multiply all volumes by the # of rxns required in that primer/probe mix (determined in 3.2). See example below. Primer/probe mix Component Initial conc. Final conc. Vol. per rxn (µl) Total volume (µl) x 26 F/R primer 50µM 500 nm Probe 20µM 200 nm dh2o n/a n/a Total Ensure there are sufficient volumes of primers and probes at the appropriate initial concentration. If not, make new aliquots from stock solutions. Standard F/R primer: 50µL of 100uM F + 50µL of 100uM R = 100µL of 50µM F/R primer. Standard probe: 20µL of 100µM probe + 80µL of dh2o = 100uL of 20µM probe. Part 4. Make master mix Choose a tube large enough to hold the calculated total volume of master mix. Ex. 1.7mL Eppendorf, 5mL Falcon, or 15mL Falcon. Vortex and spin TaqMan buffer, MgCl 2, and dntps. Invert and spin TaqGold. Add calculated total volume of reagents to master mix tube. Vortex and spin master mix. Keep on ice. Part 5. Make [master mix + cdna + dh2o] samples. Set-up one 1.7ml Eppendorf tube for each cdna sample. Label them 1-n. Briefly vortex master mix; spin down if necessary. Keep on ice. Spin down cdna samples. Add the calculated volume of dh2o to each tube (ex. 54.6µl). Add the calculated volume of master mix to each tube (ex. 168µl). Add the calculated volume of cdna to each tube (ex. 1.4µl). Vortex tubes and place on ice. 5

6 Part 6. Make primer/probe mixes Briefly vortex each primer and probe; spin down. Add the calculated volume of dh2o to each tube (ex. 93.6µl). Add the calculated volume of F/R primer to each tube in the strip (ex. 5.2µl). Add the calculated volume of probe to each tube in the strip (ex. 5.2µl), if using IDTs Refer to layout when pipetting primers and probes. Ex. Gene1 goes into first tube on orange strip, Gene2 goes into first tube on yellow strip, etc. Vortex and spin down tubes; place on ice. Part 7. Pipette [master mix + cdna + dh2o] onto 384 well plate. Briefly vortex and spin down [master mix + cdna + dh2o] samples. Use Repeater Pro to pipette 16µl of sample into each well designated for that sample. Ex. Sample 1 should be pipetted into wells A1-A3, B1-B3, C1-C3, and D1-D3. 16µl per well. Part 8. Pipette primers onto 384 well plate and run. Briefly vortex and spin down primer mixes. Use the 12.5ml Matrix Multichannel pipette to pipette primer mixes into wells. Draw up 12µl primer mix from each tube in the strip and dispense 4µl into each well within a sample, moving across the row. STOP after each sample. Discard pipette tips, replace with new tips, and repeat in the next sample. When finished, seal the plate with an optical ABI cover and spin at 2000 rpm for 1 minute. If bubbles are present, drop plate onto table or tap; re-spin if necessary. Run in ABI 7900 in the following conditions: 95 for 10 minutes; 40 cycles of 95 for 15 seconds; 60 for 1 minute. Normally the instrument is pre-set, so it is only necessary to delete the first step. If using the 7900 in the lab, uncheck 9600 emulation before running plate. 6

7 Gene1 Gene2 Gene3 Gene4 Gene1 Gene2 Gene3 Gene4 A B C D E F G H I J K L M N O P Figure 1. Standard plate layout for 16 samples and 4 genes. 7