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綠色熒光定量聚合酶鏈反應議定書

閱讀次數：2289 發布時間：2012/9/14 8:45:22

Summary

Quantitative PCR is a method used to detect relative or absolute gene expression level. All qPCR involves the use of fluorescence to detect the threshold cycle （Ct） during PCR when the level of fluorescence gives signal over the background ａnd is in the linear portion of the amplified curve. This Ct value is responsible for the accurate quantization of qPCR.

SYBR Green is a dye that intercalates with double-stranded DNA. This intercalation causes the SYBR to fluoresce. The qPCR machine detects the fluorescence ａnd software calculates Ct values from the intensity of the fluorescence.

This protocol will cover the SYBR Green quantitative PCR technique, ａnd includes suggestions about which kits to use ａnd an overview of how to analyze your data.

Preparation

Quantitative PCR was initially developed to detect the copy number of transcribed mRNA, ａnd that is basically what we still do when we perform qPCR.

1.） First, RNA must be isolated from your samples. Use a technique for isolating RNA that suits you best. TRIzol methods work well, ａnd there are many good kits for it available.

Recommended: For isolating from cell culture ａnd mouse liver, I use Qiagen’s RNeasy Mini Kit （Cat. No. 74104）

This kit will work on many tissues ａnd cell types. For aorta, I use Qiagen’s RNeasy Fibrous Tissue Mini Kit （Cat. No. 74704）

2.） In order to preserve RNA samples, which are very vulnerable to degradation at room temperature, I recommend using a first-strand DNA synthesis on your RNA in preparation for qPCR, as opposed to running RT-PCR simultaneously with qPCR.

Recommended: For first-strand DNA syntheses, I use Invitrogen’s SuperScript III First-Strand Synthesis System for RT-PCR （Cat. No. 18080-051）

This will create cDNA in a 1:1 ratio to the RNA in your sample.

3.） For the qPCR itself, you will need your cDNA samples, standards made from the samples, primers specific for your genes of interest, ａnd a SYBR Green mix （which will include SYBR Green dye, Taq Polymerase, ROX, ａnd dNTP all in one）.

Recommended: SYBR Green kits are available from many companies. I recommend Qiagen’s QuantiTect SYBR Green PCR Kit （Cat. No. 204143 for 500 rxns, Cat. No. 204145 for 2500 rxns）

You will also need an internal control as a point of comparison for your data. Choose a housekeeping gene that is endogenously expressed in your cell type （e.g. β-2 microglobulin, β-actin）.

4.） Finally, before making the plate, make sure to sign up to use a quantitative PCR machine ahead of time. Two available options:

1. You can sign up to use the one in the Genotyping Core on 5^th floor Gonda by calling x72461. The cost is $40 per plate, ａnd you need to sign in when you submit the plate.

2. Or you can use the one in Dr. Steve Young’s laboratory. Sign up online at http://calendar.yahoo.com with login “younglabqpcr” ａnd password “7500abi”.

Making the Standards

Every gene you run on qPCR will need to be run with a standard curve in order to relatively quantitate the Ct values of your samples.

The following protocol assumes that you have created cDNA from your RNA prior to qPCR. If you followed the Invitrogen SuperScript III kit’s protocol, you should start with 21μl of cDNA per sample.

1.） Dilute each cDNA sample ~4-fold. In this case, dilute your 21μl with 59μl H₂O for a final volume of 80μl. Vortex ａnd spin down.

2.） Pool an equal amount from each sample into a single tube. This will be Standard 1, your high standard. To determine how much to pull from each sample, calculate how much you will have left in each sample ａnd what the final volume of your standards will be. （This is to have approximately the same final volume of standards ａnd samples.）

Ø Example.）

Take 30μl from each of 12 samples ａnd pool for Standard 1 with a final volume of 360μl.

Final sample volume will be sample volume * 5 after a five-fold dilution （see below）. In this case, （80μl - 30μl） * 5 = 250μl of each sample, final volume.

Take 90μl of Standard 1 in a new tube labeled Standard 2. Dilute Standard 2 with 270μl H₂O for a final volume of 360μl. Repeat up to Standard 5.

Final standard volume will be initial standard volume - 90μl after making the next standard. In this case, 360μl Standard 1 - 90μl = 270μl of standard, final volume.

3.） Create the rest of your standards by taking out 1/4^th of the last standard ａnd diluting it 4-fold. Each standard will have a value assigned to it, as below.

Example.）

Standard Number	Dilution Factor	Dilutions	Value
Standard 1	Pool	360μl Standard 1	25600
Standard 2	1:4	90μl Stnd. 1 + 270μl H₂O	6400
Standard 3	1:16	90μl Stnd. 2 + 270μl H₂O	1600
Standard 4	1:64	90μl Stnd. 3 + 270μl H₂O	400
Standard 5	1:256	90μl Stnd. 4 + 270μl H₂O	100

4.） Remember to vortex ａnd spin down after each dilution step!

Samples

Dilute your samples （cDNA） further in a 1:5 dilution with H₂O. For example, dilute your remaining 50μl of sample in 200μl H₂O for a final volume of 250μl.

Making the Plate

Before making the plate, draw a layout of how you will pipette it ahead of time so you know what is in each well.

Use plates appropriate to the machine you’ll be using. For the ABI7500 Fast PCR system, use Optical 96-Well Fast Thermal Cycling Plates from ABI （Part No.: 4346906）. Use Optical Caps from ABI （Part No.: 4323032） with this plate.

Example） Two genes run on one plate. 18 samples total.

High St. 1

St. 2

St. 3

St. 4

Low St. 5

No cDNA

Gene 1

High St. 1

St. 2

St. 3

St. 4

Low St. 5

No cDNA

Gene 2

Your plate should look something like the above plate. It needs to include a standard curve for each gene being tested, ａnd needs a water （no cDNA） control for each gene. Be sure to use the correct type of qPCR optical plates ａnd caps for the specific machine you are using. A normal PCR plate will not work.

A typical plate will end up having 20μl in each well, final volume. Add samples ａnd standards in the volumes listed below.

Ø For the No cDNA wells, pipette 8μl of H₂O.

Ø For standard wells, pipette 8μl of the corresponding standard.

Ø For sample wells, pipette 8μl of the corresponding sample.

Note: Each sample, standard ａnd no cDNA control will be in duplicate as above.

After pipetting the above, create 12μl of ｍaster mix for each well, as below, plus excess.

1 rxn: 10μl SYBR Green 52 rxn: 520μl SYBR

0.4μl Forward Primer （10μM stock） 20.8μl F

0.4μl Reverse Primer （10μM stock） 20.8μl R

1.2μl H₂O 62.4μl H₂O

12μl per well （+ 8μl cDNA = 20μl final volume） 624μl F.V.

Pipette 12μl of your ｍaster mix into each well ａnd mix gently by pipetting up ａnd down a few times. Do not vortex, as shear stress can damage your enzyme.

Running the qPCR

Quantitative PCR requires that a certain type of machine capable of detecting SYBR fluorescence while performing PCR be used. （The Dr. Steve Young lab, for example, uses the ABI 7500 Fast Real-Time PCR system.）

Each system comes with different software to perform your PCR ａnd analyze your data. Refer to the user’s guide for whichever machine you are using to learn how to use it, or ask someone who knows to show you. At the end of this protocol, I will cover how to use the ABI7500 Fast Real-Time PCR system specifically.

These programs will invariably ask you to input the layout of your plate prior to running them. In addition, you will have to input your PCR conditions. Please use conditions ideal for your own primers.

Note: I design all of my primers to ideally function at about 60°C. Therefore, my PCR conditions resemble the following:

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94°C – 15 minutes （for the Qiagen mix mentioned earlier）

94°C – 15 sec.

60°C – 30 sec. 40 cycles

72°C – 30 sec.

Before running the plate, be sure to spin it down in an appropriate centrifuge to expel as many air bubbles as possible before running.

Analysis

On a basic level, all data from this type of quantitative PCR will be analyzed in a similar manner. However, depending on your experimental design, the way you analyze the data may vary. I’ll go over the basic analysis of data in a control vs. exposure type of experiment to give an example of how to analyze your data.

SYBR Green quantitative PCR machines take readings of the amount of double stranded DNA in each well at each cycle ａnd give the critical threshold （Ct） value, which represents the quantization of your product.

The Ct is a relative value. This is why every experiment needs an internal control, usually a housekeeping gene.

Always check your standard curves for a good slope ａnd R^2 value. The perfect slope would be –3.32, ａnd R^2 would be 1. If your slope or R^2 deviate from these values too much （i.e. a slope of around –4.0, or an R^2 below .9）, your primers are probably not very good.

Ø Example.）

In this experiment, we are comparing cells exposed to a certain oxidized phospholipid with cells that are not to see how this exposure effects their expression of Gene X.

Using a six-well cell culture plate, we exposed two of our wells to media （the controls）ａnd two to the oxidized phospholipid （the exposure）. We lysed the cells, collected the lysate ａnd isolated RNA, then ran a first-strand synthesis of DNA.

We then created standards from our samples just like above ａnd diluted our samples as above.

Sample 1 + 2 – Control （no oxidized phospholipid）

Sample 3 + 4 – Exposure （+oxidized phospholipid）

We’ll use the gene β-2 microglobulin （β2M） as our internal control.

Our plate layout looks something like this:

High St. 1

St. 2

St. 3

St. 4

Low St. 5

No cDNA

β2M

High St. 1

St. 2

St. 3

St. 4

Low St. 5

No cDNA

Gene X

After running in the qPCR machine, we obtain Ct values ａnd relative quantities calculated by the program after data analysis. It calculates these quantities by comparing the Ct of your samples with the standard curve’s arbitrary values.

Since we ran duplicates of each sample, the program should average them ａnd give you a quantity mean for each sample.

β2M is a housekeeping gene, so its expression should not change between control ａnd exposure. Therefore, we use it as a point of comparison for Gene X’s expression.

Say we get the following data for each sample:

Sample	β2M	Gene X
1	14000	8800
2	13800	9300
3	12200	13700
4	12400	13500

Start by dividing Gene X’s values by β2M’s values. This will establish the relative values for Gene X’s induction.

Sample	β2M	Gene X	Gene X/β2M
1	14000	8800	.629
2	13800	9300	.674
3	12200	13700	1.123
4	12400	13500	1.089

Now divide this value by the average of your control values for each one. This normalizes your control’s value to 1 ａnd allows you to see the relative induction or reduction of your exposure samples. Average your replicates to get your final value.

Sample	β2M	Gene X	Gene X/β2M	Normalization	Induction
1	14000	8800	.629	0.965	1
2	13800	9300	.674	1.035
3	12200	13700	1.123	1.724	1.698
4	12400	13500	1.089	1.672

In this example, Gene X in the experimental samples （3 ａnd 4） is induced about 70% in response to exposure to the oxidized phospholipid.

Your method of analysis depends on the type of experiment you are performing. However, you will always need to normalize your data at some point, as above, in order to obtain the relative values for your gene of interest’s change in expression.

SYBR qPCR Quick Protocol

Standards

Ø Dilute the cDNA ~4-fold （e.g. 21μl sample + 59μl H₂O = 80μl）

Ø Pool an appropriate amount from each sample to create standard 1 （i.e. 30μl x 12 samples = 360μ standard 1; Calculate out the final volume of your standards ａnd try to make it as close to the final volume of your samples as possible.）

Ø Create standards as follows

o Pool 360μl standard 1 （25600）

o 1:4 90μl standard 1 + 270μl H₂O （6400）

o 1:16 90μl standard 2 + 270μl H₂O （1600）

o 1:64 90μl standard 3 + 270μl H₂O （400）

o 1:256 90μl standard 4 + 270μl H₂O （100）

Ø Mix well at each step.

Samples

Ø Dilute the remaining samples （cDNA） further in a 1:5 dilution with H₂O. For example, dilute your remaining 50μl sample in 200μl H₂O.

Ø Your samples will end up being about 1/5^th of the high standard.

Plate

Ø For the No cDNA wells, pipette 8μl of H2O.

Ø For the standard wells, pipette 8μl of standard.

Ø For the sample wells, pipette 8μl of sample.

Ø Run each sample ａnd standard in duplicate.

Assay

Make 12μl of ｍaster mix for each well, plus some excess

1 Rxn: 10μl SYBR Green Mix 52 Rxn: 520μl SYBR

0.4μl Forward Primer （10μM stock） 20.8μl F

0.4μl Reverse Primer （10μM stock） 20.8μl R

1.2μl H₂O &n, bsp; 62.4μl H₂O

12μl per well （+ 8μl cDNA = 20μl final volume） 624μl

Analysis

（on the ABI7500 Fast Real-Time PCR System in Dr. Steve Young’s Lab）

1.） Sign up online at http://calendar.yahoo.com with login “younglabqpcr” ａnd password “7500abi”.

2.） Bring your plate up to MRL 4629. The ABI machine is in a room at the back right of 4629.

3.） Start up the computer ａnd ABI7500 machine to allow it to heat up before use.

4.） Spin down your plate in the Eppendorf centrifuge for large tubes ａnd plates just outside the ABI room. Make sure the wells are free of bubbles.

5.） Open the ABI7500 software ａnd prepare your template.

6.） Set your standard values accordingly （25600 for the high standard down to 100 for the low standard）.

7.） Set up the temperatures ａnd times for your runs accordingly. Be sure to set it to run “standard”, not “fast”. Set the volume to 20μl ａnd have it take data during the third step.

8.） Start the run.

9.） After the run, use the analysis tools to analyze your data. Set it to “Auto Ct” ａnd have the program analyze. Check your standard curves for a good slope ａnd R^2 value （the perfect slope would be –3.32, ａnd R^2 would be 1）.

10.） Analyze your data in Excel. Your method of analysis depends on your experimental design.

總結

定量聚合酶鏈反應是一種用來檢測相對或絕對的基因表達水平。所有涉及使用熒光定量PCR檢測閾值循環（電腦斷層）在反應時的水平，熒光信號的背景，給出了在線性部分的放大曲線。這個值是負責準確量化定量PCR。