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PDF电子书：RNA Purification
目录：0 `3 A5 u% q/ k5 m1 n6 ~
Selecting a Purification Strategy . . . . . . . . . . . . . . . .. . . . . . . . . 198
: F2 d1 A- S1 @( ^Do Your Experiments Require Total RNA or mRNA? . . . . . 198
. }6 J9 G- O! OIs It Possible to Predict the Total RNA Yield from
, |6 m) ^; Y- h0 m \3 R; Ka Certain Mass of Tissue or Number of Cells? . . . . . . . . 201
D( _" p- w9 a; bIs There Protein in Your RNA Preparation, and
' D8 I+ ]) j/ Q/ c0 b1 v" jIf So, Should You Be Concerned? . . . . . . . . . . . . . . . . . . . . 202
0 a- }! i _/ L, DIs Your RNA Physically Intact? Does It Matter? . . . . . . . . . . 202# T& I" g% N8 i z* q/ f3 G; `
Which Total RNA Isolation Technique Is Most
/ c! q' {3 D% s' mAppropriate for Your Research? . . . . . . . . . . . . . . . . . . . . . 203
. ?* q2 O* \0 E$ p# BWhat Protocol Modifications Should Be Used for9 p# p9 ~, y. L* i
RNA Isolation from Difficult Tissues? . . . . . . . . . . . . . . . . 207' G% r8 Z& A' s" r5 Z
Is a One-Step or Two-Step mRNA-(poly(A) RNA)-4 l+ F" f# N4 _6 \- E+ a
Purification Strategy Most Appropriate for Your
7 ^& o/ [- H1 R2 p# l$ U& [+ N. hSituation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2097 m4 s" v; g( t
How Many Rounds of Oligo(dT)–Cellulose8 Z+ C; W. o( J: a! F) E% t
Purification Are Required? . . . . . . . . . . . . . . . . . . . . . . . . . 210
! }, u F) U: t) [6 l9 XWhich Oligo(dT)–Cellulose Format Is Most: s2 ~9 H. E1 T1 a# z' a% L& L9 u
Appropriate? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210/ R" x4 t; a2 |" V6 }
Can Oligo(dT)–Cellulose Be Regenerated and Reused? . . . 211
2 k/ e: n6 V/ z9 M2 p( h7 WCan a Kit Designed to Isolate mRNA Directly from
+ G6 t5 s8 ^5 [2 ^6 uthe Biological Sample Purify mRNA from Total RNA? . . . 212 E( R3 o- y% [ Y& b* f/ S
Maximizing the Yield and Quality of an RNA Preparation . . . 212' S0 c5 |) h8 F
What Constitutes “RNase-Free Technique”? . . . . . . . . . . . . 212
5 c' _) l" J# P1 q7 G% M, FHow Does DEPC Inhibit RNase? . . . . . . . . . . . . . . . . . . . . . . 213
) q7 [6 o8 Y2 Y( ~9 E% m+ `3 aHow Are DEPC-Treated Solutions Prepared? Is
4 x* n2 n" ~" I8 WMore DEPC Better? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
: u, b1 ^+ M$ {! d1 w9 p! a4 PShould You Prepare Reagents with DEPC-Treated Water,
- U" ^9 N) l4 \3 x& z$ [1 e+ _7 Xor Should You Treat Your Pre-made Reagents with
2 r! V3 i. q& z# V) w' u" M6 IDEPC? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
' U( j% c' N8 X; }" bHow Do You Minimize RNA Degradation during Sample/ i" I) }. x8 M$ V
Collection and Storage? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
/ G: ?. Z/ ~) j' K$ IHow Do You Minimize RNA Degradation during Sample
# X; _0 r) p- Q5 Z: b- cDisruption? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215' Z" d. J. l8 l. p. Q: E. _, s/ ]
Is There a Safe Place to Pause during an RNA
7 J1 H' N8 J/ I$ h. O- gPurification Procedure? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218" Z+ {5 M4 y' _( M) ]. S
What Are the Options to Quantitate Dilute RNA
- A+ D4 N. Z" tSolutions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
; [- }6 b& }4 iWhat Are the Options for Storage of Purified RNA? . . . . . . . 219
6 O1 v6 ?3 i2 dTroubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220# M1 Q4 w! ^. F5 a
A Pellet of Precipitation RNA Is Not Seen at the End of
1 j! S% @0 t+ Mthe RNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
7 L* u! E/ y0 B' t4 d. mA Pellet Was Generated, but the Spectrophotometer
: o8 g) h% O. V, z7 J. uReported a Lower Reading Than Expected, or Zero
2 q I. ?' Q- G8 h3 ]% SAbsorbance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
( Q$ h. m& \: O TRNA Was Prepared in Large Quantity, but it Failed
9 Z h1 n% b: S( ~+ L3 C: J+ N8 Qin a Downstream Reaction: RT PCR is an& r* {3 m0 m6 C& [: }( b m w5 t
Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
l& a/ g3 E2 X% ~+ cMy Total RNA Appeared as a Smear in an Ethidum9 u4 Q4 h0 E9 l" ^# `$ }; L& q
Bromide-stained Denaturing Agarose Gel; 18S and7 d# [( _& g! J4 q2 d: H
28S RNA Bands Were not Observed . . . . . . . . . . . . . . . . 222
; g- S, c2 z" K" s: R K3 ^, FOnly a Fraction of the Original RNA Stored at -70°C) T7 Z% @, k' V5 C7 \- W- O, C& Q
Remained after Storage for Six Months . . . . . . . . . . . . . . 222* U% f0 v D- ?2 s0 |
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 |
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发表于 2011-10-25 19:26
发表于 2011-10-26 08:59
