真核细胞总RNA的制备

从细胞中分离RNA的纯度于完整性对于许多分子生物学实验至关重要。如Northern印迹与杂交分析、寡聚（dT）纤维素选择分离mRNA，cDNA合成及体外翻译等实验的成败，在很大程度上决定于RNA的质量。
RNA分离的最关键因素是尽量减少RNA酶的污染。
快速一步法提取总RNA
组织及有核细胞在匀浆过程中被变性液破膜、溶解，变性剂抑制RNA酶的活性，并使蛋白质变性及与核酸分离。经酸酚/氯仿将RNA抽提至水相，与DNA和蛋白质分离，再经异丙醇沉淀回收总RNA。
【试剂】
1×CSB缓冲液： 柠檬酸三钠 25mmol/L pH7.0
十二烷基肌苷酸钠 0.5%
b-巯基乙醇 0.1mol/L
配制100ml 5×CS缓冲液：柠檬酸三钠3.6762，十二烷基肌苷酸钠2.5g溶于100ml 0.05％DEPC水中，过滤，15lb/in2， 20min，去DEPC，4°C保存。
用前100ml工作液加 b-巯基乙醇700ml。
4mol/L异硫氰酸胍变性缓冲液：
47.26g异硫氰酸胍加至100ml CSB缓冲液中。
配制：23.63g异硫氰酸胍加热65°C溶于20ml无RNase水中，加10 ml 5×CS，加350ml b-巯基乙醇，用无RNase的水定量至50ml。（约加25ml水）
2mol/L 乙酸钠NaAc（pH4.0）：
16.4g乙酸钠加至0.05％DEPC水40ml，用冰乙酸调pH至4.0，定容至100ml，分装，高压20min去除DEPC。
3mol/L 乙酸钠NaAc (pH5.2):
24.6g乙酸钠加至0.05％DEPC水80ml，用冰乙酸调pH至5.2，定容至100ml，分装，高压20分钟去除DEPC。
水饱和酚（酸酚，pH4.0）：
取重蒸酚200ml，于65°C水浴溶解，加100ml无RNase水混匀，静置，取上层水相（大部分），加0.2g 8-巯基喹啉，混匀，保存于棕色广口瓶，4°C待用。

【操作方法】
1． 组织匀浆：新鲜的或液氮冻存的组织，称重后，按100mg组织/ml变性液，匀浆。置冰上30min。
培养细胞：贴壁细胞用PBS洗2次后，直接加2ml变性缓冲液/瓶；悬浮细胞用PBS洗沉淀2次，按107细胞/ml变性缓冲液，置冰上30min。
2． 取0.5ml粘稠液体加入1.5ml Eppendorf管（10ml塑料离心管）中，加50ml （1/10体积）2 mol/L NaAc（pH4.0），混匀。
3． 加0.5ml(等体积)酚和100ml (1/10体积的)氯仿：异戊醇（49:1），振荡混匀，冰浴10min。
4． 4°C，12000g，20min。
5． 吸上层水相入新管，加等体积异丙醇，-20°C，至少1hr。
6． 4°C，12000g，20min。
7． 弃上清，沉淀用1ml预冷的70%乙醇漂洗，并移入Eppendorf管。
8． 4°C，12000g，10min。
9． 弃上清，室温干燥蒸发残存乙醇，数分钟。
10．加100ml 水溶解RNA，测含量。
11．加1／10体积3mol乙酸钠(pH5.2)和2.5倍体积预冷的无水乙醇置-30℃30min。
12．离心，12000g，15min．
13．弃上清，加预冷的70％乙醇1ml，12000g，离心5min。弃上清，室温放置数分钟。
溶解RNA：每20mg RNA加4.5ml水溶解RNA，4℃存放待转膜（不宜久放）。

Total RNA Isolation
Guanidine-based isolation

Objective:
To obtain total RNA from zebrafish embryos.

Required Materials
Denaturing Solution or Solution "D"
2 M NaOAC pH 4
Phenol, H₂O saturated
49:1 Chloroform/Isoamyl alcohol
Isoproponal
75% EtOH
DEPC-treated H₂O or freshly deionized formamide

1 mL syringe
20 gauge needle
1.5mL microcentrifuge tubes
microcentrifuge

Procedures
Start=> Collected zebrafish embryos of desired developmental stage, etc.
Remove excess H₂O from embryos.
Using a 1mL syringe with a 20 gauge needle, add the appropriate amount (consult Table 1) of solution "D" to the embryos.
Homogenize the embryos by passage through the syringe needle (5-8) times. To decrease the volume of frothy homogenate and check the status of the embryos, briefly pulse in a microcentrifuge at low speed. If there is a pellet of embryonic tissue, continue to homogenate with the syringe needle. If not, continue with the extraction.
The homogenates can be safely stored at -80°C.
Add 2M NaOAc pH4 (consult Table 1), and mix by inversion.
Add phenol and chloroform:isoamyl alcohol, vortex, and incubate on ice for 5-10'.
Centrifuge at 14,000 rpm for 2-3' at 4°C, and transfer upper aqueous phase to a new microcentrifuge tube.
Add 100% Isopropanol to precipitate RNA. Incubate samples at -20°C for at least 30'.
Centrifuge at 14,000 rpm for 10' at 4°C, and discard supernatant in waste container.
Dissolve the RNA pellet in solution "D", and transfer 10μL to a new microcentrifuge tube.
Precipitate RNA by adding equal volumes of 100% isopropanol. Incubate samples at -20°C for at least 30'.
Centrifuge at 14,000 rpm for 10' at 4°C, and discard supernatant in waste container.
Wash pellet by adding 75% EtOH.
If RNA is to be stored, leave majority of RNA precipitated in the 75% EtOH at -80°C. Continue with the 10μL aliquot to determine approximate concentration and integrity of rRNA bands.
Centrifuge at 14,000 rpm for 10' at 4°C, and discard supernatant in waste container.
Pulse the pellet to collect remaining supernatant with a pipet tip. Allow the pellet to air dry or use a vacuum.
Resuspend in DEPC-treated H₂O or formamide.
Suspension in formamide protects the RNA from degradation by RNases, but for most applications the RNA should be precipitated from the formamide by adding 4 volumes of 100% EtOH and centrifuging at 14,000 rpm for 10' at 4°C.,
Quantitate RNA by diluting 1 μL into 100μL with alkaline H2O (see below). Then determine the A260 and A280.
Water used for spectrophotometric measurement of RNA should have a pH of > 7.5. Acidic pH affects the UV absorption spectrum of RNA and significantly decreases the A₂₆₀/A₂₈₀. Adjust water to a slightly alkaline pH by adding concentrated Na₂HPO₄ solution to a final concentration of 1mM.