(完整版)microRNA定量PCR檢測(cè)方法

1、microRNA定量PCR檢測(cè)實(shí)驗(yàn)設(shè)計(jì)首先特異性檢測(cè)：最常用的 ABI公司的Taqman探針?lè)?，其策略?采用發(fā)卡RT引 物反轉(zhuǎn)錄，隨后taqman探針做real-time。ABI的TaqMan探針?lè)?，設(shè)計(jì)的是頸環(huán)引 物，針對(duì)特定的microRNA，反轉(zhuǎn)后以特定的引物和探針做熒光定量。反轉(zhuǎn)錄引物和熒光定量引物及探針組成一個(gè) assay。大多數(shù)研究的位點(diǎn)，都能在ABI網(wǎng)站上找到現(xiàn) 成的assay。TaqMan探針?lè)z測(cè)靈敏度高，目前大多數(shù)文章中都采用的這種方法。同時(shí)TaqMan探針技術(shù)是專利技術(shù)，所以相對(duì)較貴。如果資金有限，可以使用SYBR染料法代替，這方面很多公司都有相應(yīng)的試劑盒，比如TIAN

2、GEN TaKARA等，國(guó)內(nèi)公司廣州銳博或上海吉瑪也可以，相對(duì)便宜一些。其次是非特異性方法，即總RNA加上poly A尾巴，再用poly T的引物做反轉(zhuǎn)，然后 用SYBR Gree做熒光定量。代表性的Qiagen方法是首先給 miRNA加poly( A)+adapter， 然后利用adapter的序列作為反向引物， miRNA本身為正向引物(或者 5 端修飾 下)。然后和普通real-time PCR一樣進(jìn)行就可以了。這個(gè)可以自己設(shè)計(jì)， adapter就 是一段隨即引物，末端轉(zhuǎn)移酶等。具體可以搜下相關(guān)資料。關(guān)于microRNA定量PCR的RT弓I物：1、 Oligo d(T)特異的RT引物 Q

3、IAGEN產(chǎn)品為主由特異序列 Oligo d (T)20左右 兼并堿基V或VN組成。 所有miRNA可以公用一個(gè) Oligod(T)的RT引物 但是RNA在反轉(zhuǎn)錄前需要進(jìn)行末端 Poly(A)加尾2、莖環(huán)狀結(jié)構(gòu)的 RT引物 ABI產(chǎn)品為主 由可以自身呈環(huán)莖狀的特異序列 6 到8個(gè)miRNA3端反向互補(bǔ)堿基組成。(一條miRNA序列特異對(duì)應(yīng)一個(gè)莖環(huán)狀結(jié) 構(gòu)的RT引物1) stem-loop RT引物設(shè)計(jì)：基于通用的莖環(huán)結(jié)構(gòu)，只需要按照不同的 miRNA序列 修改最末端 6 個(gè)堿基即可。通用莖環(huán)結(jié)構(gòu)序列為：GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC例如

4、設(shè)計(jì) miR-1 (UGGAAUGUAAAGAAGUAUGUA)的RT引物，只需在通用莖環(huán)序列后架上miRNA3末端的6個(gè)堿基的反向互補(bǔ)序列，即GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGAAACCATC2) realtime引物設(shè)計(jì)：上游引物，miRNA序列除去3端 6個(gè)堿基的剩余部分作為上游引物，如 miR-1的上游引物為(注意把U改為T(mén))： TGGAATGTAAAGAAG檢查弓I 物的Tm值，如果Tm值較低，則在5端加GC使Tm值接近60度(primer express軟 件計(jì)算更準(zhǔn)確)。因此 miR-1的上游引物可設(shè)計(jì)為： GCGCTGGAATGTAAAG

5、AAGTT 游引物是通用的，序列為 GTGCAGGGTCCGAGGT引 I物設(shè)計(jì)好后，需要通過(guò)預(yù)試驗(yàn) 檢測(cè)引物的特異性。SYBR染料法一般需要做溶解曲線來(lái)檢測(cè)引物的特異性；同時(shí)最好將PCR產(chǎn)物進(jìn)行電泳檢測(cè)產(chǎn)物是否單一（因產(chǎn)物長(zhǎng)度很小，需要3%以上的瓊脂糖膠）。3）探針設(shè)計(jì)（primer express）: TaqMan探針位置盡可能靠近擴(kuò)增引物 （擴(kuò)增產(chǎn)物50-150bp），但不能與引物重疊。長(zhǎng)度一般為18-40mer （最好是20-30bp）。避免連續(xù)相同堿基的出現(xiàn)，特別是要避免GGGG或更多G出現(xiàn)。在引物的5端避免使用G-因?yàn)?G會(huì)有淬滅作用，而且即使是被切割下來(lái)還會(huì) 存在淬滅作用。可選用

6、比較多的堿基Co退火溫度Tm Tm值在65-70 C,通常比引物TM值高5-10C（至少要5C） , GC 含量在40% 70%oRT primerri i iiiniirStep 1: Stemdoop RT111 limnmiRNACONAStep 2: Real-time PCRReverse primerTaqMan probe實(shí)例參考：1. RT-PCF引物 及探針設(shè)計(jì):Real-time quantification of microRNAs by stem -loop RT-PCR, Caifu Chen et al. Nucleic Acids Research, 2005,

7、Vol. 33, No. 20 e179Reverse tran scriptase react ions contained RNA samples in cludi ng purified total RNA, cell lysate, or heat-treated cells, 50 nM stem -oop 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 M

8、ultiScribe reverse tran scriptase (P/N: 4319983,Applied Biosystems) and0.25 U/ml RNase inhibitor (P/N:N8080119; Applied Biosystems). The 7.5m l reactions were in cubated 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

9、 at 4C. All Reverse tran scriptase reac-tio ns, 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 System (P/N: 4329002, Applied Biosystems). The 10ml PCR in

10、cluded 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 react ions were in cubated 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 rea

11、ctions were run in triplicate. The threshold cycle ( CT) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. TaqMa nC T values were con verted in to absolute copy nu mbers using a sta ndard curve from syn thetic lin-4 miRNA.2.血清血漿中 miRNA定量檢測(cè)方法：Analysis of

12、circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR),EvarM. Kroh et al. Methods 50 (2010) 2984301Reverse tran scripti onReverse transcription reactions are performed using the Taq-Man miRNA Reverse Transcription Kit and miRNA-specific stem-loop p

13、rimers (Part No. 4366597, Applied BioSystems, I nc.) in a scaled dow n (5IL) RT reactio n: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 pri

14、mer. 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 in put RNA. For gen erati on of sta n-dard curves using chemically s

15、yn thesized RNA olig onu cleotides corresponding to known miRNAs, serially dilute the synthetic miRNAs as described in Section 2.8 and add to RT react ions at a volume of 1.67uL per react ion.Mix RT react ions by in vers ion and cen trifuge to collect conten ts.Use a Tetrad2 Peltier Thermal Cycler (

16、BioRad) to carry out the RT react ions using the followi ng con diti ons: 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 reacti ons are performed in duplicate, in scaled-dow n (5uL

17、) react ion 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 pri

18、mer/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 suffic

19、ient 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 reacti