microRNA定量PCR检测方法.doc

microRNA 定量PCR检测实验设计

l 首先特异性检测：最常用的ABI公司的Taqman 探针法，其策略是 采用发卡RT引物反转录，随后taqman探针做real-time。ABI的TaqMan探针法，设计的是颈环引物，针对特定的microRNA，反转后以特定的引物和探针做荧光定量。反转录引物和荧光定量引物及探针组成一个assay。大多数研究的位点，都能在ABI网站上找到现成的assay。TaqMan探针法检测灵敏度高，目前大多数文章中都采用的这种方法。同时TaqMan探针技术是专利技术，所以相对较贵。如果资金有限，可以使用SYBR染料法代替，这方面很多公司都有相应的试剂盒，比如TIANGEN，TaKARA等，国内公司广州锐博或上海吉玛也可以，相对便宜一些。

l 其次是非特异性方法，即总RNA加上poly A尾巴，再用poly T的引物做反转，然后用SYBR Green做荧光定量。代表性的Qiagen方法是首先给miRNA加poly（A）+adapter，然后利用adapter的序列作为反向引物，miRNA本身为正向引物（或者5‘端修饰下）。然后和普通real-time PCR一样进行就可以了。这个可以自己设计，adapter就是一段随即引物，末端转移酶等。具体可以搜下相关资料。

关于microRNA定量PCR的RT引物：

l 1、 Oligo d(T)特异的RT引物QIAGEN产品为主 由特异序列Oligo d (T)20左右兼并碱基V或VN组成。所有miRNA可以公用一个Oligod(T)的RT引物但是RNA在反转录前需要进行末端Poly(A)加尾

l 2、茎环状结构的RT引物ABI产品为主 由可以自身呈环茎状的特异序列6到8个miRNA3’端反向互补碱基组成。(一 条miRNA序列特异对应一个茎环状结构的RT引物

1）stem-loop RT引物设计：基于通用的茎环结构，只需要按照不同的miRNA序列修改最末端6个碱基即可。通用茎环结构序列为：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC
例如设计miR-1（UGGAAUGUAAAGAAGUAUGUAU）的RT引物，只需在通用茎环序列后架上miRNA3’末端的6个碱基的反向互补序列，即
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATACAT
2）realtime 引物设计：上游引物，miRNA序列除去3’端6个碱基的剩余部分作为上游引物，如miR-1的上游引物为(注意把U改为T)：TGGAATGTAAAGAAGT.检查引物的Tm值，如果Tm值较低，则在5’端加GC使Tm值接近60度（primer express 软件计算更准确）。因此miR-1的上游引物可设计为：GCGCTGGAATGTAAAGAAGT。下游引物是通用的，序列为GTGCAGGGTCCGAGGT。引物设计好后，需要通过预试验检测引物的特异性。SYBR染料法一般需要做溶解曲线来检测引物的特异性；同时最好将PCR产物进行电泳检测产物是否单一（因产物长度很小，需要3%以上的琼脂糖胶）。
3）探针设计（primer express）：
◆TaqMan 探针位置尽可能靠近扩增引物(扩增产物50-150bp)，但不能与引物重叠。
◆长度一般为18-40mer（最好是20-30bp） 。
◆避免连续相同碱基的出现，特别是要避免GGGG或更多G出现。
◆在引物的5’端避免使用G--因为5'G会有淬灭作用，而且即使是被切割下来还会存在淬灭作用。可选用比较多的碱基C。
◆退火温度Tm Tm值在65-70℃，通常比引物TM值高5-10℃（至少要5℃），GC含量在40%－70%。

实例参考：
1. RT-PCR引物 及探针设计：

Real-time quantification of microRNAs by stem–loop RT–PCR, Caifu Chen et al. Nucleic Acids Research, 2005, Vol. 33, No. 20 e179
Reverse transcriptase reactions contained RNA samples including purified total RNA, cell lysate, or heat-treated cells, 50 nM stem–loop RT primer (P/N: 4365386 and 4365387, Applied Biosystems), 1x RT buffer (P/N:4319981, Applied Biosystems), 0.25 mM each of dNTPs,3.33 U/m l MultiScribe reverse transcriptase (P/N: 4319983,Applied Biosystems) and 0.25 U/ml RNase inhibitor (P/N:N8080119; Applied Biosystems). The 7.5m l reactions were incubated in an Applied Biosystems 9700 Thermocycler in a96- or 384-well plate for 30 min at 16C, 30 min at 42 C, 5 min at 85C and then held at 4C. All Reverse transcriptase reac-tions, including no-template controls and RT minus controls,were run in duplicate.

Real-time PCR was performed using a standard TaqMan PCR kit protocol on an Applied Biosystems 7900HT Sequence Detection System (P/N: 4329002, Applied Biosystems). The 10ml PCR included 0.67 ul RT product, 1x TaqMan Uni-versal PCR Master Mix (P/N: 4324018, Applied Biosystems),0.2uM TaqManm probe, 1.5uM forward primer and 0.7uM reverse primer. The reactions were incubated in a 384-well plate at 95C for 10 min, followed by 40 cycles of 95C for 15 s and 60C for 1 min. All reactions were run in triplicate. The threshold cycle ( CT) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. TaqManC T values were converted into absolute copy numbers using a standard curve from synthetic lin-4 miRNA.

2. 血清血浆中miRNA定量检测方法：

Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR),Evan M. Kroh et al. Methods 50 (2010) 298–301
Reverse transcription
Reverse transcription reactions are performed using the Taq-Man miRNA Reverse Transcription Kit and miRNA-specific stem-loop primers (Part No. 4366597, Applied BioSystems, Inc.) in a scaled down (5lL) RT reaction:
Each reaction should be comprised of 1.387 uLH2O, 0.5u 10 xReverse-Transcription Buffer, 0.063 uL RNase-Inhibitor (20 U/uL), 0.05u L 100 mM dNTPs with dTTP, 0.33 uL Multiscribe Reverse Transcriptase, 1uL RT primer. These components should be prepared as a larger master mix. Mix by inversion (do not vortex), and collect contents by brief centrifugation.
Aliquot master mix into 0.2 mL RNase-free striptubes or a 96-well plate and add 1.67uL input RNA. For generation of stan-dard curves using chemically synthesized RNA oligonucleotides corresponding to known miRNAs, serially dilute the synthetic miRNAs as described in Section 2.8 and add to RT reactions at a volume of 1.67uL per reaction.
Mix RT reactions by inversion and centrifuge to collect contents.Use a Tetrad2 Peltier Thermal Cycler (BioRad) to carry out the RT reactions using the following conditions: 16 C for 30 min,42C for 30 min, 85 C for 5 min, hold at 4 C. RT products can be stored undiluted at 20 C prior to running the real-time PCR.

Real-time PCR
Real-time PCR reactions are performed in duplicate, in scaled-down (5uL) reaction volumes using 2.5uL TaqMan 2 x Universal PCR Master Mix with No AmpErase UNG, 0.25uL miRNA-specific primer/probe mix, and 2.25uL diluted RT product per reaction.
1. For each miRNA-specific assay, prepare a reaction pre-mix by combining sufficient TaqMan 2x Universal PCR Master Mix and primer/probe mix for all reactions, plus excess for losses associated with pipetting. Mix by inversion and centrifuge briefly.
2. Aliquot enough reaction mix for two duplicate reactions per sample into striptubes (i.e., 5.5uL reaction mix plus 15% excess for pipetting losses, per aliquot, will be sufficient for duplicate reactions for a sample, because 2.75 u L reactin pre-mix is needed per final reaction).
3. Dilute the RT products by combining 5.0uL RT product with 28.9uL water (1:15 final dilution in PCR reaction). Mix and centrifuge briefly.
4. For each sample, add the diluted RT product to the reaction premix aliquots from step 2, adding enough for duplicate reactions (i.e., 4.5 lL diluted RT product plus 15% excess, because 2.25uL diluted RT product is needed per PCR reaction). Mix the duplicate reactions, centrifuge, and aliquot 5uL reactions in duplicate into the optical plate. Seal with ABI MicroAmp™ Optical
Adhesive Film. Centrifuge plate to ensure no bubbles inhibit signal detection.
5. Real-time PCR is carried out on an Applied BioSystems 7900HT thermocycler (Applied Biosystems, Inc.) using the following conditions: 95 C for 10 min, followed by 40 cycles of 95 C
for 15 s and 60 C for 1 min, followed by a hold at 4 C. Raw data can then be analyzed with SDS Relative Quantification Software version 2.2.3 (Applied BioSystems, Inc.), generally using the automatic cycle threshold (Ct) setting for assigning baseline and threshold for Ct determination.