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？

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

Yedan

http://www.targetscan.org/
7 Y4 |) j2 {% J这是根据基因预测mir的" U* D7 d4 [  y; h5 g
http://cbcsrv.watson.ibm.com/rna22.html
- L8 P( m3 r$ l) JRNA22这是根据sequence预测的 包括CDS和3’UTR

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。& w6 N. t" }7 `* v2 k, O- R, l# }3 M
http://www.targetscan.org/

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miRNA
9 B* u) C$ B) J+ O" TAmbion, Invitrogen

telomerase

Designing miR-Mimics' G. G6 F. K2 T7 H( R: G/ m, p
1. Defining a unique sequence in the target gene. A fundamental requirement to
; R( \; X! I. U1 `0 }. Z5 F4 u6 Mbe satisfied is that the 3UTR of the target gene must contain a unique sequence4 y0 i. X! `& }0 \1 F
distinct from other genes to elicit gene-specific action. Similar to designing
' F- {' t8 r, f& S! A1 Ia siRNA, the first step to design a miRNA mimic is to identify a stretch of
- I9 j) j  x, D. W7 {sequence in the 3UTR unique to the gene of interest (target mRNA). But5 S( X/ m) j2 J- k6 b% `
unlike the full complementarity between a siRNA and its target in any region of" e9 [& q( ~3 U9 F9 c3 M8 K
the gene, a miRNA mimic partially base-pairs with the target sequence in the
, Y; z( O+ @$ Y4 V3UTR. The length of the sequence should be long enough for miRNA action,
  T  u! u5 `7 `6 I' T" twhich is at least 8 nts and ideally >14 nts;
3 v: ?/ @  R' N" e0 p3 _2 g0 G2. Based on the unique sequence, design a 22-nt oligonucleotides that at the 51 U# ?& @7 E7 C9 o) f
portion has 8 nts (nucleotide positions 1–8 from 5-end) fully complementary. h, v3 m$ F7 P- V
to the target mRNA;/ x7 D% V0 ~% h5 x5 _4 K) P
3. To ensure the specificity of binding to the target mRNA, the oligonucleotides( t1 X8 d( G* ~' v' q* }/ t
should have at least additional 5–6 nts complementary to the target mRNA at
5 d1 U  t) Z2 k+ Mthe 3 portion. The base-pairing in this region may not necessarily be continuous4 `; |) y% k8 E  F) `
and can be grouped into 3-nts clusters;( C/ k% Y; ?9 @: W* W# c# B
4. Add an “AU” to the3 end of the fragment. As such, a 22-nt miR-Mimic should! y# j  _& L8 z3 q8 j- c8 Z
have at least 14 complementary nucleotides to the target gene plus an “AU”5 K' @/ j+ ?8 C( g& i
overhand;
2 K1 J* e  ^6 }# d' p* h5. Design an antisense strand fully complementary to the fragment;
; A+ w2 O5 |: L' C+ N6. Both of the two oligonucleotide fragments need to be artificially synthesized.
6 k( y/ a) [' O  L" M5 {+ qMany companies provide excellent services for nucleic acids research, such as6 l0 r' K; [/ C* M$ n' w
Integrated DNA Technologies, Ambion, Invitrogen, etc;( s2 V; [8 v; |9 C( k
7. Anneal the sense and antisense strands of the oligonucleotides to form a duplex
2 z8 F( r; |0 E9 V! imiR-Mimic. First mix the two synthetic oligonucleotides at an equalmolar concentration/ j( W3 d! X% W8 j
in an Eppendorf tube, then incubate the sample in a heat block at
. j' j0 |; P7 F/ D4 z- }4 ^1 C95◦C for 5 min and gradually cool the sample to room temperature (22–23◦C);( ^! l; _) B5 x1 o: u
8. Store the miR-Mimic construct at –80◦C for future use;
8 J9 k/ e% V9 A7 k. m) {$ w0 e9. Construct a negative control miR-Mimic (NC miR-Mimic) for verifying the) ]$ L0 H2 _7 v0 Z
effects and specificity of the effects of the miR-Mimic. This NC miR-Mimic" p! e; Q: M2 c' P, Y/ u  C2 y
should be designed based on the sequence of the miR-Mimic; simply modifying
6 }: o, L3 i+ C( E- q4 Wthe miR-Mimic sequence to contain ∼5-nt mismatches at the 5-end “seed site”.' H6 a$ z) M! T) q" Y! E
Such modified or mutated miR-Mimic is expected to lose the ability to bind to" r0 ]) U% r! f2 B3 T
the target mRNA with sufficient affinity and is thus rendered incapable of eliciting9 t* b% n! f4 s- Z0 b) [
repressive action. To form an NC miR-Mimic, the annealing procedures; M1 K5 m, l" F, H, D8 G
described above need to be repeated;
" M$ E2 O( L4 }6 D3 z3 g! K. E10. Synthesize an anti-miRNA inhibitor (AMO) against the miR-Mimic as an, K0 J) X$ ^1 s# c* Q4 w
additional negative control. An AMO is designed to be an exact antisense to
1 N- C0 w: c% J( s* G2 _& Uits target miR-Mimic. An AMO is a single-stranded oligonucleotide (ON) or- o+ |! [) O8 b' n. D0 R- x
oligodeoxynucleotide (ODN) fragment (see detail in Chap. 7);
  w0 C; N+ V( M+ O- h' `+ W11. As for SC-miRNAs, chemical modification should be done for miR-Mimic.
* j# [% g: P8 W& q! aThe type of modification should be determined based on your particular need: x* M* w+ E& A; S2 i3 D
(Fig. 4.1).