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传送门：mRNA疫苗质量分析程序-歪把子.pdf
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Cyclesequencing: Remove the seal from the plate and add 2 µL dye sequencing mastermix, 1 µL of dye tagged forward, and tagged reverse primer【19】.
[NOTE—Addtagged forward primer to one of the duplicate PCR reactions, and the taggedreverse primer to the other reaction.]
Seal theplate, vortex the mixture briefly, and centrifuge for 5–10 s at 1000 x g. Putsamples in the thermal cycler and run the program as detailed in Table 4.
循环测序:
[注释-在重复的PCR反应中加入标记的正向引物，在另一个反应中加入标记的反向引物。]
封板，将混合物短暂涡旋，1000 × g离心5-10 s。将样品放入热循环器中，运行程序如表4所示。
Table 4:Thermal Cycler Conditions (Cycle Sequencing)
表4:热循环器条件(循环顺序)
Sequencingclean-up: Centrifuge the reaction plate for 1 min at 1,000 x g. Prepare a mixtureaccording to the kit. There are several kits available to support removal ofunincorporated terminators and salts.20
测序清理:用1000 x g离心反应板1分钟。根据试剂盒准备混合物。有几种工具包可用于支持去除未合并的终止剂和盐【20】。
[NOTE—Makesure solutions are homogeneous with no particulates before using.]
【注意使用前确保溶液均匀，无颗粒。】
Add 55µL of this mixture to each well. Seal the plate, vortex the reaction plate for40 min, and centrifuge the plate for 2 min at 1000 x g.
向每孔中加入55升这种混合物。封板，涡旋反应板40分钟，1000 x g离心2分钟。
Collectionof data: Load the plate into the genetic analyzer such as the capillaryelectrophoresis (see integrity methods below). Select or create an appropriaterun module according to capillary length, number of capillaries, and polymertype on the instrument. The electrophoresis will separate the labeledchain-terminated fragments by length with single-nucleotide resolution. Oncethe run is finished, the instrument will generate a file that can be convertedinto a sequence.
数据收集:将培养皿装入基因分析仪，如毛细管电泳(见下面的完整性方法)。根据毛细管长度、毛细管数量和仪器上聚合物类型选择或创建合适的运行模块。电泳将以单核苷酸长度的分辨率，分离标记的链终止片段。一旦运行完成，仪器将生成一个文件，该文件可以转换成一个序列。
Dataanalysis: Use a sequence scanner software to generate a report. Software shouldbe able to call low frequency somatic variants at a detection level below 5%.
数据分析:使用序列扫描软件生成报告。软件应该能够在低于5%的检测水平上调用低频体细胞变异。
Method C: Identity by RT-PCR
方法C:RT-PCR鉴定
Reversetranscription PCR (RT-PCR) can be used to identify and quantify mRNA and isperformed in two steps: reverse transcription (first strand of cDNA synthesis),and PCR amplification.
10 mMdNTP mix: Mix 10 mM of each nucleotide (dATP, dCTP, dGTP and dTTP) in 0.6 mMTris-HCl.
Firststrand buffer (5X): Mix 250 mM of Tris-HCl at pH 8.3, 375 mM of KCl, and 15 mMof MgCl2.
PCRbuffer (10X): 200 mM of Tris HCl at pH 8.4 and 500 mM of KCl.
Firststrand cDNA synthesis: Prepare the following mixed solution.
逆转录聚合酶链反应(RT-PCR)是对mRNA进行鉴定和定量的一种方法。逆转录聚合酶链反应分为两步:逆转录(cDNA合成的第一链)和PCR扩增。
10mMdNTP混合:将每种核苷酸(dATP、dtp、dGTP和dTTP)各10mM混合在0.6 mM Tris-HCl中。
第一链缓冲液(5X):在pH 8.3下混合250 mM的Tris-HCl,375 mM的KCl，和15 mM的MgCl2。
PCR缓冲液(10倍):200 mM的TrisHCl pH 8.4和500 mM的KCl。
cDNA第一链合成:制备以下混合溶液。
Table 5:First-Strand cDNA Solution表5:第一链cDNA 溶液
Heat themixture at 65° for 5 min and then quickly cool on ice for 2 min. Centrifuge for5–10 s at 1,000 x g. Next, prepare a reverse transcription reaction system bycombining the following solutions.
将混合物在65℃下加热5分钟，然后在冰上快速冷却2分钟。用1000x g离心5 10 s。然后，将以下溶液组合在一起，制备一个逆转录反应系统。
Table 6:Reverse Transcription Reaction Solution
Gentlyvortex the mixture for few minutes. If random primers are used, incubate at 25° for 2 min, then add 1 μL (200 U) of Reverse Transcriptase to thereaction tube and mix gently with pipette. Incubate at 42-50° for 50 min.
轻轻地搅拌几分钟。如果使用随机引物，25°孵育2 min，然后在反应管中加入1μL (200 U)的逆转录酶，以移液管轻轻混合。42 - 50°孵育50分钟。
[NOTE If reverse primer of PCR is used as areverse transcription primer, it is recommended to perform the reaction at45-50° ; otherwise, it is generally recommended toperform the reaction at 42° .]
【注:若采用PCR的下游引物作为逆转录引物，建议反应温度为45 ~ 50°;否则，一般建议在42°时进行反应。]
Inactivate and stop the reverse transcriptionreaction by heating at 70° for 15 min. Sample can beused immediately for subsequent PCR reactions or can be stored at -20° for short-term storage and -80° forlong-term storage.
70℃加热15min，使逆转录反应失活并停止。样品可立即用于后续的PCR反应，也可保存在-20℃短期保存，-80℃长期保存。
RT-PCR:Using Table 7 prepare a 50 μL reaction solution.
RT-PCR:根据表7制备50 μL反应液。
Table7: RT-PCR Reaction Solution 表7:RT-PCR反应溶液
[NOTE—Thereare 4 different fluorescent DNA probes that are available for RT-PCR productdetection. These products are SYBR Green, TaqMan, Molecular Beacons andScorpions. All these probes allow the detection of PCR products by generating afluorescent signal. Follow manufacturers’ protocols for each.]
【注：有4种不同的荧光DNA探针可用于RT-PCR产物检测。这些产品分别是SYBR Green, TaqMan, Molecular Beacons和Scorpions。所有这些探针都可以通过产生荧光信号来检测PCR产物。遵循每种产品的制造商协议。】
Gentlymix the reaction and place it in the thermal cycler using the followingprogram.
缓缓地混合以使反应，并将其放入热循环器中，按以下程序运行。
Table 8:Thermal Cycler Conditions (First Strand Synthesis) 表8:热循环器条件(第一链合成)
Preparationof cDNA for standard curve: Standard curve is necessary to quantitate theresults. Dilute stock plasmid 1:1000 to a dilution of 1 ng/μL. Preparestandards as described below.
标准曲线的制备:标准曲线是定量结果的必要条件。原液质粒1:1000稀释至1 ng/μL。准备如下所述的标准。
[NOTE—Avoidusing a plasmid that contains a gene of interest to avoid contamination.]
[注意:避免使用含有感兴趣基因的质粒以避免污染。]
Table 9:cDNA Standard Curve Preparation 表9:cDNA标准曲线制备
Quantitation 定量
Method A: Quantitation by digital PCR 方法A:数字PCR定量法
DigitalPCR can be used for mRNA quantification without a standard curve. Genomic RNAis reverse transcribed to cDNA and amplified, followed by quantitation on adigital or droplet digital PCR System.【21】
数字PCR可以用于无标准曲线的mRNA的定量。基因组RNA逆转录为cDNA并扩增，然后在数字或液滴数字PCR系统上定量。
10 mMdNTP mix:
Firststrand buffer (5X):
Primer/probemix (20X):
Firststrand cDNA synthesis:
[NOTE—To increase the efficiency of cDNAsynthesis, the reverse transcription reaction should include a targetgene-specific primer that is the same primer used as reverse primer for eachtarget in the ddPCR reaction.]
【注:为了提高cDNA合成效率，逆转录反应应包括一个目标基因特异性引物，该引物与ddPCR反应中每个目标的逆转录引物相同。】
Table10: First-Strand cDNA Solution 表10:第一链cDNA 溶液
Heat themixture at 65° for 5 min, and then quickly cool on ice for 2 min. Centrifugefor 5–10 s at 1,000 x g. Next, prepare a reverse transcription reaction systemby preparing the following mixed solution.
将混合物在65°下加热5分钟，然后在冰上快速冷却2分钟。1000× g离心5 - 10 s。然后，通过制备以下混合溶液，制备逆转录反应体系。
Table11: Reverse Transcription Reaction Solution表11:逆转录反应溶液
Gentlyvortex the mixture for few seconds. If random primers are used, incubate at 25° for 2min then add 1 μL (200 U) of reversetranscriptase to the reaction tube and mix gently with pipette. Incubate at 42–50° for 50 min.
轻轻地搅拌几秒钟。如果使用随机引物，25°孵育2 min后，在反应管中加入1 μL (200 U)的逆转录酶，与移液管轻轻混合。42-50°孵育50分钟。
[NOTE—If reverse primer of PCR is used as areverse transcription primer, it is recommended to perform the reaction at 45–50°, otherwise, generalrecommendation is to perform the reaction at 42°.]
【注-如果使用PCR的下游引物作为逆转录引物，建议反应温度为45-50°，否则一般建议反应温度为42°。】
Inactivateand stop the reverse transcription reaction by heating at 70° for 15min. Sample can be used immediately for subsequent PCR reactions or can bestored at –20° forshort-term storage and –80° forlong-term storage.
70°加热15min，使反转录反应失活并停止。样品可立即用于后续的PCR反应，也可在-20°短期保存，-80°长期保存。
Expression by dPCR:
dPCR表达:解冻所有组分，包括引物/探针混合。此外，底漆/探针混合也可以购买【22】。通过涡旋器，以最大的速度，每管30秒，确保混合彻底。简单离心收集底部的内容物。在冰上准备以下反应混合物。
Table12: dPCR Reaction Mixture表12:dPCR反应混合物
以最大速度旋转每根管子10秒，彻底混合。简单离心，让反应管平衡到室温，时间不超过10分钟。
Dropletgeneration:
液滴生成:将上述每种反应混合物各20 μL装入DG8弹的样品孔中【23】。加70 μL微滴发生油到专为“油”设计的油筒的底端行。安装橡胶DG80垫片到药筒上，并将其放在液滴发生器上。这个过程大约需要1分钟。液滴停留在最上面的一排。用多通道移液器将45 μL液滴转移到96孔PCR板上，并立即用箔片覆盖。用PCR封板器在180度下封板5秒。
Run theplate on thermocycler using the following cycling conditions.
使用以下循环条件在热循环器上运行96孔板。
【【译者：Dropletgeneration 这一节实在没接触过，几个文献地址列出来自行查看吧
https://www.bio-rad.com/sites/default/files/webroot/web/pdf/lsr/literature/10026235.pdf
https://www.bio-rad.com/sites/default/files/webroot/web/pdf/lsr/literature/10033173.pdf
https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10000050421.pdf
https://www.bio-rad.com/sites/default/files/webroot/web/pdf/lsr/literature/10000050420.pdf
https://bio-rad-sds.thewercs.com/DirectDocumentDownloader/Document?prd=HRLS00192-2~~PDF~~MTR~~CGHS~~CN
https://www.youtube.com/watch?v=GB4wcQsCawU
https://www.youtube.com/watch?v=WU3qKhIUc54
https://www.youtube.com/watch?v=VxQQx9comX0
https://www.youtube.com/watch?v=_fYQWAL7xWE】】
Table13: Thermal Cycler Conditions表13:热循环器条件
[NOTE—To determine acceptable temperature ranges for reverse transcription, perform athermal gradient from 42° to 51.5° while fixing the annealing/extension step at 52°. Using the optimized reverse transcription temperature, perform a thermalgradient from 50° to 63° toidentify acceptable annealing/extension temperature ranges.]
[注-为确定逆转录可接受的温度范围，需在42°至51.5°之间进行热梯度，同时将退火/延伸步骤固定在52°。利用优化的逆转录温度，进行50°至63°的热梯度，以确定可接受的退火/扩展温度范围。]
Dataanalysis:
数据分析:根据所使用的系统，按照说明进行数据采集和分析。
