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 o 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TIA

2、NGEN, 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 Gree儆荧光定量。代表性的Qiagen方法是首先给 miRNA力口 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 M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弓I物：1) Oligo d(T)特异

3、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2) 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：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGA

4、TACGAC例如设计 miR-1 (UGGAAUGUAAAGAAGUAUGUAU的RT弓I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GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGAAACCAC3) 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

5、： GCGCTGGAATGTAAAGAAG仆 游引物是通用的，序列为 GTGCAGGGTCCGAGGTI物设计好后，需要通过预试验 检测引物的特异性。SYB戏料法一般需要做溶解曲线来检测引物的特异性；同时最3%以上的琼脂好将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

6、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Co 退火温度 Tm Tm值在65-70 C,通常 比引物TM值高5-10 C （至少要 5C） , GC 含量在40%70%。miRNART primerI lllllllStep 1:Stem-loo口 RTCONA1 I I I I I I I I I I I I lllllllStep 2:Real-time PCRForward primer0TaqMan probeReverseprimer实例参考：1 . RT-PC咫I物 及探针设计:Real-time quantification of microRNAs by s

7、tem Hoop RT-PCR, Caifu Chen et al.Nucleic Acids Research, 2005, Vol. 33, No. 20 e179Reverse 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:4319

8、981, Applied Biosystems), 0.25 mM each of dNTPs,3.33 U/m l MultiScribe reverse transcriptase (P/N: 4319983,Applied Biosystems) and0.25 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

9、 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 PCRkit protocol on an Applied Biosystems 7900HT Sequence Detection Sy

10、stem (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 b

11、y 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 synthe

12、tic 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),EvaM. Kroh et al. Methods 50 (2010) 298 W01Reverse transcriptionReverse transcription reactions are performed using the Taq-Man miRNA Reverse Tr

13、anscription 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 Mu

14、ltiscribe 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

15、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 Tetr

16、ad2 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 PCRReal-time PCR reactions are performed in duplicat

17、e, 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 P

18、CR 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 ali

19、quot, 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 reac