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PDF电子书：RNA Purification
目录：
9 S: ~# D0 K4 y: Q* W( _Selecting a Purification Strategy . . . . . . . . . . . . . . . .. . . . . . . . . 198% h* p9 y' e$ x$ j. I; a
Do Your Experiments Require Total RNA or mRNA? . . . . . 198
0 P8 U' T. v+ S6 r& HIs It Possible to Predict the Total RNA Yield from* ?! i: h* B" b- w$ r
a Certain Mass of Tissue or Number of Cells? . . . . . . . . 201: s1 u' k$ o+ \" u, b r
Is There Protein in Your RNA Preparation, and. U8 K% \/ g9 j1 X l) W
If So, Should You Be Concerned? . . . . . . . . . . . . . . . . . . . . 202/ a" b% n5 k X+ A" h- U& D
Is Your RNA Physically Intact? Does It Matter? . . . . . . . . . . 202+ V, U6 L& |' \1 M- ?
Which Total RNA Isolation Technique Is Most5 c0 M* {- x! W6 Y* Z7 y* l# p
Appropriate for Your Research? . . . . . . . . . . . . . . . . . . . . . 203* _, G7 l1 ^+ K& v S1 x7 b$ P
What Protocol Modifications Should Be Used for
, @1 m T/ z8 j7 q4 HRNA Isolation from Difficult Tissues? . . . . . . . . . . . . . . . . 2071 u& g9 a$ i0 l$ A2 q) d) |7 _
Is a One-Step or Two-Step mRNA-(poly(A) RNA)-8 f5 ~* h+ X" K( u6 }" V8 y
Purification Strategy Most Appropriate for Your! A8 E- ~$ U7 z
Situation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
' s0 H- d1 }, `& w Y3 g0 GHow Many Rounds of Oligo(dT)–Cellulose( Q! Z0 M4 g. Z
Purification Are Required? . . . . . . . . . . . . . . . . . . . . . . . . . 210
# `; J" ^/ d: L5 O, q/ S$ D* qWhich Oligo(dT)–Cellulose Format Is Most& C( @9 O/ |/ g& \( U. W! [9 r
Appropriate? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210* A. k+ _2 _2 S( f
Can Oligo(dT)–Cellulose Be Regenerated and Reused? . . . 2111 G d6 C6 ~% w2 {
Can a Kit Designed to Isolate mRNA Directly from; I R R4 i. f
the Biological Sample Purify mRNA from Total RNA? . . . 212
% Z2 s8 Q( z- W- L* N5 aMaximizing the Yield and Quality of an RNA Preparation . . . 212$ c) B3 v! r, v/ t
What Constitutes “RNase-Free Technique”? . . . . . . . . . . . . 212/ J/ f' Q* \2 v. L
How Does DEPC Inhibit RNase? . . . . . . . . . . . . . . . . . . . . . . 213
) D7 s1 x e* }8 o; M/ C7 \How Are DEPC-Treated Solutions Prepared? Is
/ X$ B: ^; T9 t; q) ~More DEPC Better? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
$ z3 c& `# U/ MShould You Prepare Reagents with DEPC-Treated Water,+ G- Q! v2 Q4 o/ x
or Should You Treat Your Pre-made Reagents with- w- B7 ^$ t6 s- J g0 U
DEPC? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
* `- {# @" w8 M. r1 c" h! G# {How Do You Minimize RNA Degradation during Sample& E% {6 W' I) |# A
Collection and Storage? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
2 l! x/ D' {! t' C( DHow Do You Minimize RNA Degradation during Sample
# ^% a! t% p, T2 b! ZDisruption? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215, ]7 x& Z, ~' s- u7 R4 o- P$ I
Is There a Safe Place to Pause during an RNA7 V2 w7 [6 o: C) Y) a
Purification Procedure? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 ]- H- J6 S1 ^* ]: |) M- H) @! T
What Are the Options to Quantitate Dilute RNA
$ A: m: F: a/ \! Q; Q2 u7 B3 zSolutions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218* F, F) p' Y1 N# E
What Are the Options for Storage of Purified RNA? . . . . . . . 219
9 L8 u9 z, c7 q; B+ O+ k* X8 f1 JTroubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
: P3 z2 [# j$ N1 ]. KA Pellet of Precipitation RNA Is Not Seen at the End of
' U$ M% ^1 r: w. r# `the RNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220- G0 ^* [+ {6 e: b8 l# ]
A Pellet Was Generated, but the Spectrophotometer
0 z U w7 Z+ _$ `: zReported a Lower Reading Than Expected, or Zero1 Y! D2 Y# G+ R5 l- T, a* O
Absorbance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2211 Z+ K) k, r8 N1 ^+ t& }- H& Q) ?% c
RNA Was Prepared in Large Quantity, but it Failed; s$ S2 F6 M$ j* o( z& e1 e( T; D
in a Downstream Reaction: RT PCR is an
4 [ s2 ^) k' {2 u& MExample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221* X- \. W, a; C3 u& y
My Total RNA Appeared as a Smear in an Ethidum/ ?8 P* c7 a8 r: ?
Bromide-stained Denaturing Agarose Gel; 18S and: G* s) Z" B6 l `" ?
28S RNA Bands Were not Observed . . . . . . . . . . . . . . . . 2224 _& }! k# f$ x% Q; ]
Only a Fraction of the Original RNA Stored at -70°C
. D3 G5 f8 t1 NRemained after Storage for Six Months . . . . . . . . . . . . . . 222
) r4 _2 I! F) S* S. y) x7 N; D, CBibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 |
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发表于 2011-10-25 19:26
发表于 2011-10-26 08:59
