(完整版)microRNA定量PCR检测方法

1、microRNA 定量 PCR检测实验设计首先特异性检测：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

2、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。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

3、定量 PCR的 RT引物：1、 Oligo d(T)特异的 RT引物兼并碱基 V 或 VN 组成。RNA在反转录前需要进行末端QIAGEN产品为主由特异序列 Oligo d (T)20 左右所有 miRNA 可以公用一个Oligod(T)的 RT 引物但是Poly(A)加尾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-loopRT 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：GTCGT

4、ATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC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GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGATACATC2）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 T

5、m 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 20-30

6、bp） 。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 stemloop RTPCR, Caifu Chen et al. Nucleic Acids Research, 2005, Vol. 33, No. 2

7、0 e179Reverse transcriptase reactions contained RNA samples including purified total RNA, cell lysate, or heat-treated cells, 50 nM stemloop 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

8、transcriptase (P/N: 4319983,Applied Biosystems) and0.25 U/ml RNase inhibitor (P/N:N8080119; Applied Biosystems). The 7.5m l reactions wereincubated in an Applied Biosystems 9700 Thermocycler in a96- or 384-well plate for 30 minat 16C, 30 min at 42 C, 5 min at 85C and then held at 4C. All Reverse tra

9、nscriptase 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

10、, 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 triplica

11、te. 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：AnalysisofcirculatingmicroRNA biomarkersinp

12、lasmaandserumusingquantitative reverse transcription-PCR (qRT-PCR),EvanM. Kroh et al. Methods 50(2010) 298301Reverse transcriptionReverse transcription reactions are performed using the Taq-Man miRNA Reverse Transcription Kit and miRNA-specific stem-loop primers (Part No. 4366597, Applied BioSystems

13、, 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 l

14、arger 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 kno

15、wn 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 cond

16、itions: 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 PCRReal-time PCR reactions are performed in duplicate, in scaled-down (5uL) reaction volumesusing 2.5uL TaqMan 2 x Universal PCR Master Mix wi

17、th 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 TaqMan2x Universal PCR Master Mix and primer/probe mix for all reactions, plus excess for losses associated with

18、 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-

19、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, cen