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绿色荧光定量聚合酶链反应议定书
点击次数：2610发布时间：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 5th 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 1/4th 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.
| | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | High St. 1 | St. 2 | St. 3 | St. 4 | Low St. 5 | No cDNA | 1 | 2 | 3 | 4 | 5 | 6 | |
|
Gene 1 B | " | " | " | " | " | " | " | " | " | " | " | " | |
| C | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | |
| D | " | " | " | " | " | " | " | " | " | " | " | " | |
| E | High St. 1 | St. 2 | St. 3 | St. 4 | Low St. 5 | No cDNA | 1 | 2 | 3 | 4 | 5 | 6 | |
|
Gene 2 F | " | " | " | " | " | " | " | " | " | " | " | " | |
| G | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | |
| H | " | " | " | " | " | " | " | " | " | " | " | " |
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:
| | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | High St. 1 | St. 2 | St. 3 | St. 4 | Low St. 5 | No cDNA | 1 | 2 | 3 | 4 | | | |
|
β2M B | " | " | " | " | " | " | " | " | " | " | | | |
| C | | | | | | | | | | | | | |
| D | | | | | | | | | | | | | |
| E | High St. 1 | St. 2 | St. 3 | St. 4 | Low St. 5 | No cDNA | 1 | 2 | 3 | 4 | | | |
|
Gene X F | " | " | " | " | " | " | " | " | " | " | | | |
| G | | | | | | | | | | | | | |
| H | | | | | | | | | | | | |
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 1/5th 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.
总结
定量聚合酶链反应是一种用来检测相对或绝对的基因表达水平。所有涉及使用荧光定量PCR检测阈值循环（电脑断层）在反应时的水平，荧光信号的背景，给出了在线性部分的放大曲线。这个值是负责准确量化定量PCR。
绿色荧光是一种染料，也是与双链脱氧核糖核酸。插导致荧光的荧光。该定量PCR检测荧光和软件计算值从荧光强度。
这个协议将包括绿色荧光定量聚合酶链反应技术，包括建议，工具使用和概述了如何分析你的数据。
制备
定量聚合酶链反应最初制定检测拷贝数的转录表达，基本上就是我们还是当我们进行定量PCR。
1。）第一，核糖核酸是孤立从样品。使用的技术隔离核糖核酸，最适合你。提取的方法工作，并有许多好的工具包，它提供。
推荐：孤立的细胞培养和小鼠肝，我使用的试剂试剂盒试剂盒（猫。74104号）
这包将在许多组织和细胞类型。主动脉，我使用的试剂试剂盒纤维组织迷你包（猫。74704号）
2）。为了保存样品，这是非常容易降解在室温下，我建议使用一个第一链合成的核糖核酸聚合酶链反应制备，而RT - PCR同时运行。
建议：为第一链脱氧核糖核酸合成，使用我公司的标三第一链合成系统的RT - PCR（猫。第18080-051）
这将创建在一个1 : 1的比例，在您的样品。
3。）的定量PCR本身，你将需要你的基因样本，从样本的标准，具体的引物的基因的兴趣，和荧光绿色混合（其中包括绿色荧光染料，耐热聚合酶，火箭，和核苷酸都在一个）。
建议：绿色荧光试剂盒可从许多公司。我建议试剂盒的quantitect绿色荧光聚合酶链反应试剂盒（猫。204143号的500 rxns，猫。204145号的2500 rxns）
你还需要一个内部控制点的比较数据。选择管家基因，是内源性表达的细胞类型（例如β- 2微球蛋白，β-肌动蛋白）。
4。）最后，前板，确保注册使用定量聚合酶链反应机提前时间。2可用选项：
1。您可以使用注册一个在基因型芯第五楼所有的x72461。成本是40美元，而你需要登录时提交板。
2。或者你可以使用一个在史提夫博士的实验室。网上报名在http://calendar.yahoo.com登录“younglabqpcr“密码”7500abi”。
制定标准
每一个基因上运行的定量PCR将需要运行一个标准曲线，相对定量值的样品。
以下协议假定您已创建的核糖核酸基因PCR之前。如果你遵循公司上标三包的协议，你应该开始与21μ我每样基因。
1。）每个基因样本~ 4倍稀释。在这种情况下，稀21μ升59升的水μ最后一卷80μL涡和自旋向下。
2。）池同等数量从每个样品到一个单一的管。这将是标准的1，你的高标准。确定有多少拉从每个样品，计算出多少钱，你会把每个样品和最终体积的标准将。（这是有大致相同的最终体积的标准和样品。）
Ø例子。）
30μ从每个样品和12池标准1的最后一卷360μL
最终样本量将样品数量* 5后，5倍稀释（见下）。在这种情况下，（80 - 30μμ长）* 5 = 250μ我每个样品，最后一卷。
90μ信用标准1在一个新的管标记的标准2。稀释标准2与270μ升的水的最后一卷360μ属重复标准5。
最后的标准量将最初的标准量- 90μ我之后的下一个标准。在这种情况下，360μ标准1 - 90μ= 270μ信用标准，最后一卷。
3）创建。其余的标准采取了1 /第四的最后标准和稀释4倍。每个标准都有一个值分配给它的，如下。
例子。）
标准编号稀释因子稀释值
标准1池360μ标准1
25600
标准2
4
90μ我加戒备。1 + 270μ我水
6400
标准3
16
90μ我加戒备。2 + 270μ我水
1600
标准4
64
90μ我加戒备。3 + 270μ我水
400
标准5
256
90μ我加戒备。4 + 270μ我水
100
4。）记得涡和自旋下来后，每个稀释步骤！
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