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-Mimics
) K% J3 H) {9 Q, z5 P1 Q9 \1. Defining a unique sequence in the target gene. A fundamental requirement to' n& J7 H) w; Z9 v5 u8 Z
be satisfied is that the 3UTR of the target gene must contain a unique sequence! J7 V% t# f3 [* M/ v* \' a/ n
distinct from other genes to elicit gene-specific action. Similar to designing
7 C  U7 H" _4 l! }a siRNA, the first step to design a miRNA mimic is to identify a stretch of
. P9 ?: }& s, D7 }5 P! @, {sequence in the 3UTR unique to the gene of interest (target mRNA). But
2 M. T* R+ t0 W* iunlike the full complementarity between a siRNA and its target in any region of' ^2 O5 s2 b4 m$ _( D3 S6 \( e
the gene, a miRNA mimic partially base-pairs with the target sequence in the
! B/ r. Z/ ]& I0 e, F/ n3UTR. The length of the sequence should be long enough for miRNA action,% Q6 Z* V  N5 N2 W0 s
which is at least 8 nts and ideally >14 nts;; R* L& R* t* X( l; `
2. Based on the unique sequence, design a 22-nt oligonucleotides that at the 5
: G, r$ v5 W3 g8 g/ ^( o, kportion has 8 nts (nucleotide positions 1–8 from 5-end) fully complementary
8 \9 ~9 z- z( S/ K! Qto the target mRNA;
" I, S0 V  e7 F% Z: Q# @0 k% V3. To ensure the specificity of binding to the target mRNA, the oligonucleotides( Y7 @0 G6 O7 Y5 H0 k, x
should have at least additional 5–6 nts complementary to the target mRNA at! S3 T# B8 N; V  p6 z; g
the 3 portion. The base-pairing in this region may not necessarily be continuous# Y, V8 j$ Q! |: `
and can be grouped into 3-nts clusters;
2 I+ Q' o$ Z# Q* O2 M4. Add an “AU” to the3 end of the fragment. As such, a 22-nt miR-Mimic should( ]9 S$ w. ~/ L3 \/ A0 i5 m
have at least 14 complementary nucleotides to the target gene plus an “AU”2 e4 N; k/ Y: ~! Y& W
overhand;
$ K$ l: s. t' F- ?, Z7 k5. Design an antisense strand fully complementary to the fragment;+ c& R% A0 }& Z* t- ~* y2 ?/ g
6. Both of the two oligonucleotide fragments need to be artificially synthesized., w7 l' Q5 b3 b: m$ t- G
Many companies provide excellent services for nucleic acids research, such as
/ q/ {* P# p$ m9 h7 D8 nIntegrated DNA Technologies, Ambion, Invitrogen, etc;
# `9 D. ]! `; F, I! |" v/ \7. Anneal the sense and antisense strands of the oligonucleotides to form a duplex% V. F8 H( W7 w3 C' p7 p  W0 D# J! B
miR-Mimic. First mix the two synthetic oligonucleotides at an equalmolar concentration
/ b( g7 n: G& y; \" w1 \; q( jin an Eppendorf tube, then incubate the sample in a heat block at
3 o( b- ~: o4 R5 }& q* _95◦C for 5 min and gradually cool the sample to room temperature (22–23◦C);1 ^* J8 c3 e) D/ T! P) ?3 L8 M6 l+ Y+ Z
8. Store the miR-Mimic construct at –80◦C for future use;) a7 d1 Z; M7 ]5 O$ V; m# P. l$ v
9. Construct a negative control miR-Mimic (NC miR-Mimic) for verifying the2 i  p2 D: I, q" [" r
effects and specificity of the effects of the miR-Mimic. This NC miR-Mimic
1 h( e+ v) \3 X9 }6 o5 \6 M3 Wshould be designed based on the sequence of the miR-Mimic; simply modifying- m+ w/ ^  ^9 _- c
the miR-Mimic sequence to contain ∼5-nt mismatches at the 5-end “seed site”.2 c5 H. m( m7 q1 Q/ G
Such modified or mutated miR-Mimic is expected to lose the ability to bind to. N" Q( A: [* l3 U1 ]8 @  m) h& d  _
the target mRNA with sufficient affinity and is thus rendered incapable of eliciting# P5 \% p3 l" w7 z! v. W
repressive action. To form an NC miR-Mimic, the annealing procedures
" e) v, U$ O3 u4 U1 b! u6 M( `described above need to be repeated;+ j" o+ S5 e" |0 L! e+ n$ x! ]+ x
10. Synthesize an anti-miRNA inhibitor (AMO) against the miR-Mimic as an
% _- z7 W9 `# badditional negative control. An AMO is designed to be an exact antisense to* R0 |- y( g. {8 K2 }9 \' f' u
its target miR-Mimic. An AMO is a single-stranded oligonucleotide (ON) or" w5 r4 _( m, T: P5 r" G& y. o6 E
oligodeoxynucleotide (ODN) fragment (see detail in Chap. 7);
: _' y4 M; B8 j& M; d9 [6 |' Y4 o8 E11. As for SC-miRNAs, chemical modification should be done for miR-Mimic.1 Y0 R0 K" L& u$ f" w( m
The type of modification should be determined based on your particular need
/ X/ k/ E0 E  ?6 @) O* g  F9 t(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miRNA0 u" I8 |  t$ ?/ \* x3 }" M
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。
5 n  P3 S1 M7 N' q9 ]$ lhttp://www.targetscan.org/

Yedan

http://www.targetscan.org/0 ^0 T: z4 H+ i* W$ \' ?' X
这是根据基因预测mir的
$ y4 Z  W* g1 \' y7 Q" Phttp://cbcsrv.watson.ibm.com/rna22.html. D) W& A# Z4 v0 }( q
RNA22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