求助-如何筛选mirna

lamprr

如果我想预测某个基因和mirna之间的关系怎么办？能不能预测一个可能与此基因相关的mirna？

telomerase

Designing miR-Mimics1 v+ `0 X1 Q- m9 r. S( h; d# h+ `# b
1. Defining a unique sequence in the target gene. A fundamental requirement to( R9 H; V% q4 \5 k" _3 L& ~
be satisfied is that the 3UTR of the target gene must contain a unique sequence
6 x* X# t* x) A" K; a! j0 Wdistinct from other genes to elicit gene-specific action. Similar to designing& h+ W: t; O* E0 Q7 y
a siRNA, the first step to design a miRNA mimic is to identify a stretch of
8 l( `+ C2 s  R/ ssequence in the 3UTR unique to the gene of interest (target mRNA). But
" g& V' o; Q+ U. s2 Iunlike the full complementarity between a siRNA and its target in any region of9 g" }* ?3 s  R; ~% k$ M+ F/ g
the gene, a miRNA mimic partially base-pairs with the target sequence in the% [$ P0 d' k" _4 L7 e
3UTR. The length of the sequence should be long enough for miRNA action,5 p+ L  @- b( D% E) I
which is at least 8 nts and ideally >14 nts;
7 L5 V' v/ O" H5 w1 ~" W9 X1 c2. Based on the unique sequence, design a 22-nt oligonucleotides that at the 5
% G( N7 m3 Z5 b; n9 ?# hportion has 8 nts (nucleotide positions 1–8 from 5-end) fully complementary4 Z1 w# \# h  Y$ \
to the target mRNA;- e7 a2 I+ w$ h3 ^+ h3 F2 `
3. To ensure the specificity of binding to the target mRNA, the oligonucleotides
2 }9 Y5 X+ o! [  H$ @  }# M- gshould have at least additional 5–6 nts complementary to the target mRNA at/ I# h) x; X8 i# l" G
the 3 portion. The base-pairing in this region may not necessarily be continuous
; W" m0 p2 n$ ~: }  l' e$ Q; sand can be grouped into 3-nts clusters;
4 J4 u% I3 P' V& ~. ~+ }3 ~4. Add an “AU” to the3 end of the fragment. As such, a 22-nt miR-Mimic should
$ s: y3 f( U* |; M0 V1 ^( ^have at least 14 complementary nucleotides to the target gene plus an “AU”% S2 D4 [& }0 |5 h8 m; `# p
overhand;
7 m! D/ k  n/ U0 o" H, \* ~; q5 V5. Design an antisense strand fully complementary to the fragment;
7 Q: F: ^1 K, x5 @. i$ h2 u/ C7 W6. Both of the two oligonucleotide fragments need to be artificially synthesized.4 e1 E9 M  l  @2 g: c. d/ M; O
Many companies provide excellent services for nucleic acids research, such as
! i6 V6 ?" a% S0 A% s* d) D6 |Integrated DNA Technologies, Ambion, Invitrogen, etc;2 e. W6 F2 A2 \' ?! R
7. Anneal the sense and antisense strands of the oligonucleotides to form a duplex
" n- s3 _+ m# l6 r5 ~5 CmiR-Mimic. First mix the two synthetic oligonucleotides at an equalmolar concentration
. c: L, q0 T! ^+ ]$ H( }) I" Vin an Eppendorf tube, then incubate the sample in a heat block at
/ G0 y2 Z( m1 _" u. L5 i0 P95◦C for 5 min and gradually cool the sample to room temperature (22–23◦C);
  F1 k, P2 L' g8. Store the miR-Mimic construct at –80◦C for future use;
% B/ ~3 U3 N3 D3 P9. Construct a negative control miR-Mimic (NC miR-Mimic) for verifying the
8 i# ?. A4 r9 s, k  leffects and specificity of the effects of the miR-Mimic. This NC miR-Mimic6 e5 o, x: P6 W6 k* E
should be designed based on the sequence of the miR-Mimic; simply modifying) M' ]$ E% S$ Y* p) m' Z- T
the miR-Mimic sequence to contain ∼5-nt mismatches at the 5-end “seed site”.' g% t) P$ w4 \! ^6 E
Such modified or mutated miR-Mimic is expected to lose the ability to bind to/ h  i- @# |7 O. x  d
the target mRNA with sufficient affinity and is thus rendered incapable of eliciting
7 r! F" f: R/ k, U3 Q, Crepressive action. To form an NC miR-Mimic, the annealing procedures$ k5 J' Q* K5 d
described above need to be repeated;
+ y: B1 H4 R! h+ t4 F2 a! d10. Synthesize an anti-miRNA inhibitor (AMO) against the miR-Mimic as an
3 |+ l6 @9 h. W% w/ L! iadditional negative control. An AMO is designed to be an exact antisense to6 J7 x" ^; t/ ?
its target miR-Mimic. An AMO is a single-stranded oligonucleotide (ON) or) D5 N" A! K0 T, o" z% w) _  m! P) j
oligodeoxynucleotide (ODN) fragment (see detail in Chap. 7);
0 ^$ k4 z( k7 u7 \- F; B! Y' {& p11. As for SC-miRNAs, chemical modification should be done for miR-Mimic.$ O* U3 F/ F! M4 v$ Y4 ^
The type of modification should be determined based on your particular need: U1 C+ f7 j! ~6 d0 n2 W) I
(Fig. 4.1).

telomerase

从protocol来看，何siRNA类似，可以通过提交序列在某些网站上预测可能的miRNA3 @% `! L% ]" b8 r' p
Ambion, Invitrogen

kastanie

如果仅仅是初步预测一下的话，可以考虑computational prediction，有很多选择，比如TargetScan。
) f+ {% `& e. G7 phttp://www.targetscan.org/

Yedan

http://www.targetscan.org/
# i2 ]$ i2 u, I0 N8 D" d7 Y6 D5 F6 e3 U这是根据基因预测mir的
, D% `9 F3 }" O' bhttp://cbcsrv.watson.ibm.com/rna22.html
; O& |% k7 h5 J- R& O2 i9 ZRNA22这是根据sequence预测的 包括CDS和3’UTR

susanliuxue

miRanda、Targetscan、PicTar可以预测miRNA和miRNA之间的关系，可以试试看，我没有试过，文献这么讲的