Real-Time Quantitative PCR

Chris Tachibana July 2006

• for the Young lab MJ Research Chromo4 machine (computer name is "RTPCR" no administrative password but logon is the usual)

• Opticon Monitor 3 software installer is on the desktop, okay to copy to other computers or can download software from website (along with most recent manual)

• our rep: Dominic Andrada 800-876-3425 x 1570 dominic_andrada@bio-rad.com (call several times, leave several messages, if desperate call Richard Kurtz (Manager) x 8418 richard_kurtz@bio-rad.com) or from Jim Earlie, jim_earlie@bio-rad.com

• reagents:

- ABI 4367660 Power SYBR Green PCR Master Mix, 50 ml $1620, aliquot and store at 4o near antibodies and western blot solutions

- Invitrogen 11733046 Plat SYBR Green QPCR $500 for 12.5 mls with quote 522874-c (Morris lab's): stored in 0.5 ml aliquots, -20 enzyme freezer

- Bio-Rad 223-9441 96-well microplates, natural (clear)thin-wall (we've also used) Bio-Rad MLL-9651 Low Profile 96-well Microplates (free sample) or HSP9601 white with clear wells hard-shell

- Bio-Rad MSB-1001 Microseal “B” Adhesive Film sealers ($100 for 100)

- Bio-Rad iScript one-step RT-PCR kit SYBR Green 170-8893 200x50µl reactions

• generally, all reactions done in duplicate or triplicate; all should show similar cycle thresholds c(T)

• ref: DNA/RNA Real-Time Quantitative PCR brochure from PE Biosystems; BBRC294:347 (2002); for quantitation of ChIP DNA Steger et al. Sci 2003 299:114, Nucleic Acids Res. 2001 May 1; 29(9):E45

1. ChIP DNA standard curve

• try input DNA at 1:5, 1:25, 1:125, 1:625 dilutions, 5µl per well

• for genomic DNA, try 5 ng, 1ng, 200 pg, 40 pg per 20µl reaction (but it depends on the primers)

• for cDNA, see Lynn's protocol

2. for ChIP DNA

• for samples that gave a good signal at 5µl of 1:30 or 1:40 in a regular 20µl PCR reaction (i.e. when run on a gel, you can see an ADH2 band but no ACT1 band in Adr1-ChIP), use somewhere between 2µl straight or 5µl of 1:30 per 20µl reaction (again, depends on primers)

3. for primers:

• see Primer Design.doc for protocol from Morris lab

• current working primer combinations for ChIP DNA: ACS1 CTO A+B or D+E; ADH2 Q1 + CTOF, J or L; CTO FBP1 A+B; CTO JEN1 C+D; ICL2 F+R; COT MDH2 A+B; YIL057c F+R; ADY2 F2+R2; CTO ICL1 A+B; TEL 55 + TEL56

• see the upright -70 for our oligos for RNA (cDNA)

• small amplicon is recommended (100-200bp)

• use at 0.3 µM final

4. sample prep

• write up a master chart of well contents (for example on the template at the end of this document)

• set up the protocol and plate files in the Opticon monitor program (see next steps)

• put a 96-well plate in a plastic holder (optional: on ice)

• at the no template bench, make a master mix of, for example, water, 2X SYBR mix and primers and add to wells (20µl total reaction volume per well for ChIP DNA; 10µl per well for cDNA from RNA)

• at your bench, add the template, using a new filter pipet tip for each well

• seal the plate with an adhesive sealer. don't touch the top with your fingers. seal completely, especially around the edges

• spin in centrifuge with 96-well plate adaptors, Morris or Davis lab , for a few minutes at 1.5-2K rpm. check from the bottom for bubbles

5. Opticon machine and computer

• turn on machine in back, few minutes to warm up

• in Opticon Monitor program, go to your user (e.g. user Chris)

• files are plate, protocol, data and master

• hit save whenever making a change

• it's okay to analyze data while a run is occurring but don’t hit the red X. go back to the desktop and re-open Opticon Monitor. you'll get a message that says the machine is already in use. hit okay, go to file and open your data file.

6. plate files

• select clear or white plates

• select dye (SYB) for SYBR green

• click on individual wells (the circles), columns or rows, then label as empty, sample, blank or quantitation standard

• label wells and hit return after typing in the label

• if they are standards, hit the "specify quantitation standards" button and fill in the information (this can be done or changed after the run)

• to have replicates averaged together at the end, put them together into a set (this can can be done after the run), by selecting the replicate wells, then over on the right, selecting "sets" and naming the replicate wells

• save

7. protocol

• set reaction volume (20µl)

• use the options to write a program kind of like this

94^o 10’

94^o 25” (seconds)

60^o 25”

72^o 45” 40 cycles (=go to line 2 for 39 more cycles)

plate read

melting curve 65^o-95^o read every 0.2 seconds

72^o 10’ optional, recommended if you’re running the samples on a gel afterwards

hold at 4^o forever

(1^o sec or default ramp times)

• can do a temp gradient to find optimal annealing temp; use the “gradient calculator” function to see temps, because they are not linear across the plate

• save

8 . run

• start using "run"

• during the run, pause holds at the step it was on

• during the run, skip finishes the cycles it is on and skips the next

• to see status, hit status; to see data collected so far, hit quantitation

9. analysis

• under pulldown file menu, choose open data file

• to quantitate samples using a standard curve, group the sample and standard wells using the "manage" button under "groups" at the top of the page (hit "new", select wells to group, name them, hit "apply")

• at left sidebar, choose quantitation or melting curve

• select wells to see data or melting curve, choose either graphs or calculation with the bar at the bottom

• subtract background:

a) average over cycle range: set the baseline area to subtract, for example if there is only background fluorescence in cycles 3-8, then put those in the cycle range boxes

b) minimum over cycle range: the minimum within each reaction(?)

c) global minimum: background that is subtracted is the lowest place on the curve

• set threshold: in log scale, choose manual, and set threshold above background so it crosses all curves at the exponential range (many labs just use a lab standard consistent setting on every run)

• note the step that is shown (it indicates a read at high or low temp, for multi-read protocols)

• set background threshold (defaults at about -1.5 fluorescence)

• to save and print, go to pulldown menu quantitation menu, choose copy to clipboard, either data graph, standards graph or quant calculations

• open Excel file and paste, then email to yourself or move to another computer via the lab server

• replicates: if you "use replicates" in quantiation and calculation, then Ct is the average of points, avg Ct is the average of the merged lines

tidbits and hints

• linear range (in tests with plasmid DNA) from 10¹ to 10¹⁰ copies, Ct of 2 - in the 30s. Range of Ct's that are considered significant and linear is up to 35 or 40.

• reaction efficiencies are efficiency per well in the linear range of the Ct and two points above (so it depends on where the threshold is set)

• if comparing multiple plates, put a normalizing well with the same sample, same primers on all plates

• to keep track of the wells while you're pipetting in the template, get a new box of 20µl tips. use the tips in the same order that you fill the wells, so if you get distracted, you can look back at your pipet box and see where you left off.

• if using clear wells, look from the bottom when you're done and make sure they all appear to have the same amount

ChIP DNA Quantitation

method 1: Steger et al

• make a standard curve of input DNA in 5X dilution increments for each primer pair (assign random numbers and units, like 100, 20, 4, 0.8)

- use this as the standard for all runs and run a standard curve for each primer pair on each plate

• run ChIP samples with 1-2µl per reaction and input samples with 0.5µl-1µl per reaction for each strain or timepoint or condition

• use the standard curve to generate a number in randomly assigned units for ChIP and input samples for internal control like ACT1 or TEL and for experimental like ADH2

• calculation is [ChIP/input] for ADH2 / [ChIP/input] for TEL

• e.g. input ChIP

ADH2 50 50

ACT1 50 2

gives ADH2 increase of [50/50] / [2/50] = 1/0.04 = 25X

• note: Dave does not do replicates of ChIP or input samples on the RT-PCR plate but does experimental replicates of preps, like 3 different ChIP preps of the same condition, I think

method 2: Pfaffl

Efficiency = 10 ^(-1/slope)

max is 2 (doubling each time) and min is 1

for some reason, number of 1.935 = efficiency of 93.5%

ratio = (E target) ^{∆Ct target (e.g. input-ChIP)}

(E reference) ^{∆Ct reference (e.g. input-ChIP)}