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綠色熒光定量聚合酶鏈反應(yīng)議定書

閱讀次數(shù)：2304 發(fā)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:

}

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(jié)

定量聚合酶鏈反應(yīng)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(huán)（電腦斷層）在反應(yīng)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(zé)準(zhǔn)確量化定量PCR。

綠色熒光是一種染料，也是與雙鏈脫氧核糖核酸。插導(dǎo)致熒光的熒光。該定量PCR檢測熒光和軟件計算值從熒光強度。

這個協(xié)議將包括綠色熒光定量聚合酶鏈反應(yīng)技術(shù)，包括建議，工具使用和概述了如何分析你的數(shù)據(jù)。

制備

定量聚合酶鏈反應(yīng)*初制定檢測拷貝數(shù)的轉(zhuǎn)錄表達，基本上就是我們還是當(dāng)我們進行定量PCR。

1。），核糖核酸是孤立從樣品。使用的技術(shù)隔離核糖核酸，*適合你。提取的方法工作，并有許多好的工具包，它提供。

推薦：孤立的細胞培養(yǎng)和小鼠肝，我使用的試劑試劑盒試劑盒（貓。74104號）

這包將在許多組織和細胞類型。主動脈，我使用的試劑試劑盒纖維組織迷你包（貓。74704號）

2）。為了保存樣品，這是非常容易降解在室溫下，我建議使用一個鏈合成的核糖核酸聚合酶鏈反應(yīng)制備，而RT - PCR同時運行。

建議：為鏈脫氧核糖核酸合成，使用我公司的標(biāo)三鏈合成系統(tǒng)的RT - PCR（貓。第18080-051）

這將創(chuàng)建在一個1 : 1的比例，在您的樣品。

3。）的定量PCR本身，你將需要你的基因樣本，從樣本的標(biāo)準(zhǔn)，具體的引物的基因的興趣，和熒光綠色混合（其中包括綠色熒光染料，耐熱聚合酶，火箭，和核苷酸都在一個）。

建議：綠色熒光試劑盒可從許多公司。我建議試劑盒的quantitect綠色熒光聚合酶鏈反應(yīng)試劑盒（貓。204143號的500 rxns，貓。204145號的2500 rxns）

你還需要一個內(nèi)部控制點的比較數(shù)據(jù)。選擇管家基因，是內(nèi)源性表達的細胞類型（例如β- 2微球蛋白，β-肌動蛋白）。

4。）*后，前板，確保注冊使用定量聚合酶鏈反應(yīng)機提前時間。2可用選項：

1。您可以使用注冊一個在基因型芯第五樓所有的x72461。成本是40美元，而你需要登錄時提交板。

2。或者你可以使用一個在史提夫博士的實驗室。網(wǎng)上報名在http://calendar.yahoo.com登錄“younglabqpcr“密碼”7500abi”。

制定標(biāo)準(zhǔn)

每一個基因上運行的定量PCR將需要運行一個標(biāo)準(zhǔn)曲線，相對定量值的樣品。

以下協(xié)議假定您已創(chuàng)建的核糖核酸基因PCR之前。如果你遵循公司上標(biāo)三包的協(xié)議，你應(yīng)該開始與21μ我每樣基因。

1。）每個基因樣本~ 4倍稀釋。在這種情況下，稀21μ升59升的水μ*后一卷80μL渦和自旋向下。

2。）池同等數(shù)量從每個樣品到一個單一的管。這將是標(biāo)準(zhǔn)的1，你的高標(biāo)準(zhǔn)。確定有多少拉從每個樣品，計算出多少錢，你會把每個樣品和*終體積的標(biāo)準(zhǔn)將。（這是有大致相同的*終體積的標(biāo)準(zhǔn)和樣品。）

Ø例子。）

30μ從每個樣品和12池標(biāo)準(zhǔn)1的*后一卷360μL

*終樣本量將樣品數(shù)量* 5后，5倍稀釋（見下）。在這種情況下，（80 - 30μμ長）* 5 = 250μ我每個樣品，*后一卷。

90μ信用標(biāo)準(zhǔn)1在一個新的管標(biāo)記的標(biāo)準(zhǔn)2。稀釋標(biāo)準(zhǔn)2與270μ升的水的*后一卷360μ屬重復(fù)標(biāo)準(zhǔn)5。

*后的標(biāo)準(zhǔn)量將*初的標(biāo)準(zhǔn)量- 90μ我之后的下一個標(biāo)準(zhǔn)。在這種情況下，360μ標(biāo)準(zhǔn)1 - 90μ= 270μ信用標(biāo)準(zhǔn)，*后一卷。

3）創(chuàng)建。其余的標(biāo)準(zhǔn)采取了1 /第四的*后標(biāo)準(zhǔn)和稀釋4倍。每個標(biāo)準(zhǔn)都有一個值分配給它的，如下。

例子。）

標(biāo)準(zhǔn)編號稀釋因子稀釋值

標(biāo)準(zhǔn)1池360μ標(biāo)準(zhǔn)1

25600

標(biāo)準(zhǔn)2

90μ我加戒備。1 + 270μ我水

6400

標(biāo)準(zhǔn)3

90μ我加戒備。2 + 270μ我水

1600

標(biāo)準(zhǔn)4

90μ我加戒備。3 + 270μ我水

400

標(biāo)準(zhǔn)5

256

90μ我加戒備。4 + 270μ我水

100

4。）記得渦和自旋下來后，每個稀釋步驟！

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