Real time quantitative PCR

By Raymond Cunningham,2014-04-08 21:14
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Real time quantitative PCR

Real time quantitative PCR

Facilities and materials TM1. iCycler iQ

    2. Thin Wall PCR plate (Bio-Rad, cat. 223-9441)

    3. Optical Quality sealing tape (Bio-Rad, cat. 223-9444)

    4. Pure Dye Calibration Solution (Bio-Rad, cat. 170-8792, free by request)

    5. External well factor plate solution (Bio-Rad, cat. 170-8794, free by request)

    6. SYBR Green (Molecular Probes, cat. S-7567)


Prepare gene-specific primer sets

    1. Select genes or sequences for Real time quantitative PCR.

    2. Use the web-based primer design program called ‘Primer3’ to design primer sets

    specifying a PCR product of about 100 bp.

    3. Synthesize the designed primers.

    4. Prepare 50x primer stock solution (10 pmol/ul).

    Calibrate iCycler machine (for SYBR Green or/and other fluoresceins, but it has been done for SYBR green I, so you may skip this procedure)

    1. Pipet 50 ul of the pure dye calibration solution into 10 wells of the PCR plate.

    2. Repeat up to 3 more calibration solutions (for different fluorescent dyes).

    3. Cover the plate with sealing firm.

    4. For intercalator detection you can prepare of calibration solution of 500 pg/ul

    DNA and a 1:100,000 dilution of SYBR Green or 1 ug/ml ethidium bromide.

    5. Switch on the machine including the attached camera for 30 minutes before

    detection starts.

    6. If you are the first user after the machine is installed in a new place, logon the

    machine, open iCycler program, go to ‘Imaging Service’ by pull-down menu in

    the ‘Run Time Central Module’.

    7. Select a right filter, and an expected exposure time (minutes). Make sure the

    camera is on. Click the ‘Exposure’ on the right side of the image window. If the

    mask is not well aligned with the well fluorescent images, click ‘Align Masks’ on

    the bottom of the image window, then save it by clicking ‘Save’ in left bottom


Prepare samples for standard curves

    1. In principle, for each gene or sequence used, you need prepare a series of

    dilutions with known molecule copy number as for making a standard curve.

    2. Once obtaining the primer sets, amplify the corresponding fragments using

    common PCR machine 1) to make sure all primers are working, and 2) to have the

    PCR products suitable for preparing according standard curves.

    3. Use UV spectrometer to determine DNA concentration of the PCR products that

    have been purified through Qiagen spin columns.

    4. You may use either the actual DNA concentration (eight per volume), or copy

    number of the DNA molecule calculated based on its molecular weight. 5. Based on our experience, the dynamic range of standard preps for iCylcer is from

    2,000 to 20,000,000 copies, which can be extrapolated into a few copies.

    Total cellular RNA extraction and cDNA preparation

    1. Culture cells under desired condition.

    2. Collect cells at desired time point. TM3. Extract total cellular RNA from the cells using Ambion RNA Trizol kit.

    4. Treat RNA samples with DNase I, and purified them with Qiagen RNAeasy kit. 5. Prepare cDNA from 3-5 ug of total cellular RNA by reverse transcription:

    Total RNA 3

    Random primers 1

    dd HO 2 2

     6 ul

    Once ready, heat at 70C for 10 min, then place on ice for a while, followed by


    5x reverse tran. Buf. 2

    0.1 M DTT 1

    dNTP 0.5

    reverse transcriptase 0.5

     10 ul

    Once ready, incubate it at RT for 10 min, then at 42C for 1-2 h. Finally terminate

    it by heating at 65C for 5-10 min. Dilute 5x, and store at 40C for late use.

    Real time PCR (using SYBR Green)

    1. Prepare the experimental PCR reactions of 50 ul in a 96-well Thin Wall PCR

    plate. The recipe is as following:

    dd HO 37 2

    10x PCR buffer 5

    25 mM MgCl 3

    10 mM dNTP 0.5

    BSA 0.5

    DMSO 0.5

    cDNA 1

    Primers 1

    2,000x SYBR 1

    Taq polymerase 0.5

     50 ul

    Once ready, place on ice, and be aware to prevent light by wrapping it with

    aluminum foil.

    2. Prepare External well factor plate. If SYBR green I is used, dilute External well

    factor plate solution by a 1:10 in PCR buffer, and load 50 ul into each well of the

    96-well thin wall plate. Cover with the optical quality sealing tape. Also prevent

    light by wrapping it with aluminum foil.

    3. Use ‘Imaging service’ as before to confirm that 1x well factor solution gives a

    strong, but not saturated image.

    4. Create and save the PCR program in the ‘Protocol Workshop’. ‘Edit Protocol’ –

    in this window you may specify the thermal parameters such as followings:

    Step 1: 95C 30 sec 1 cylce

    Step 2: 95C 15 sec

    55C 30 sec

    72C 30 sec 40 cycles

     Step 3: 95C 60 sec

     Step 4: 55C 10 sec 80 cycles for melting curve

     Step 5: 4C hold forever

5. Create and save the plate setup in ‘Protocol Workshop’. “Edit Plate Setup’ –

    allows you to specify the positions of your test samples and standard samples.

    You must mark wells with specific fluorophores such as FAM-490 for SYBR

    green I. You also must key in the quantity and units (weight or copy number) for

    each individual standard well.

    6. Check PCR program and plate setup in the ‘View Plate Setup’ tab before running.

    7. Check if the machine, the camera, and the filter you selected are properly

    switched on or in the appropriate position.

    8. Place the well factor plate with the external well factor plate solution, select a

    right PCR program and a right plate setup, and click ‘Run’.

    9. In the ‘Run Prep’ tab, confirm once again the desired protocol and plate setup file.

    Enter the reaction volume (50 ul). Indicate the type of protocol (PCR

    Quantification/Melt curve) and the Well Factor Source, then click ‘Begin Run’.

    10. In about 5 min, the iCycler will go into Pause mode. During the pause period,

    you remove the well factor plate (which is transferred to 80C freezer for reuse

    late), replaced it with the experimental PCR plate. Then click ‘Continue Running


    11. After data collection on the PCR reaction plate begins, the PCR Amp Cycle plot

    will be displayed and the software will open the data analysis module.

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