Technique / Molecular Biology / RNA transcriptional post-transcriptional regulation / RNA extraction and purification

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit
(Jove (Jia Sheng Su1, Edwin S. Monuki2 1Department of Developmental and Cell Biology, University of California, Irvine, 2Department of Pathology, University of California, Irvine) RNA extraction protocol (University of Georgia) RNA extraction protocol using Trizol (Takao))
Video protocol of total RNA extraction

Total RNA and mRNA protocols
(Curr Protoc Mol Biol.)
1: Curr Protoc Mol Biol. 2002 May;Chapter 4:Unit 4.1.

Preparation of cytoplasmic RNA from tissue culture cells.

Gilman M.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

The protocol described in this unit is a fast and streamlined procedure for
preparing total cytoplasmic RNA from many cultures simultaneously for nuclease
protection analysis. It is scaled for small cultures--1 to 2 dishes of adherent
cells or 10 to 20 ml of a suspension culture. The procedure works well for many
cell types. If full-length RNA is required, ribonuclease inhibitors should be
added to the lysis buffer (as described in this unit) or the guanidinium
isothiocyanate method should be used. Finally, if RNA is isolated from
transiently transfected cells, a is provided for the treatment of RNA with
deoxyribonuclease to remove transfected DNA. This modification is especially
critical if the RNA is to be assayed by nuclease protection using uniformly
labeled probes.

2: Curr Protoc Mol Biol. 2001 May;Chapter 4:Unit4.5.

Preparation of poly(A)+ RNA.

Kingston RE.

Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts,
USA.

Most messenger RNAs contain a poly(A) tail, while structural RNAs do not. Poly(A)
selection therefore enriches for messenger RNA. The technique has proved
essential for construction of cDNA libraries. It is also useful when analyzing
the structure of low-abundance mRNAs. Removing the ribosomal and tRNAs from a
preparation increases the amount of RNA that can be clearly analyzed by S1
analysis, for example, thus allowing detection of a low level message. This
protocol separates poly(A)+ RNA from the remainder of total RNA, which is largely
rRNA and tRNA. Total RNA is denatured to expose the poly(A) (polyadenylated)
tails. Poly(A)-containing RNA is then bound to oligo(dT) cellulose, with the
remainder of the RNA washing through. The poly(A)+ RNA is eluted by removing salt
from the solution, thus destabilizing the dT:rA hybrid. The column can then be
repeated to remove contaminating poly(A)- RNA.

3: Curr Protoc Mol Biol. 2001 May;Chapter 4:Unit4.4.

Preparation of bacterial RNA.

Reddy KJ, Gilman M.

State University of New York, Binghamton, New York, USA.

Procedures for isolating RNA from bacteria involve disruption of the cells,
followed by steps to separate the RNA from contaminating DNA and protein. Lysis
strategies differ in the protocols presented in this unit, including chemical
degradation of gram-negative cell walls using sucrose/detergent or lysozyme, and
sonication to break open gram-positive cell walls. Combinations of enzymatic
degradation, organic extraction, and alcohol or salt precipitation are employed
in the procedures to isolate the RNA from other cellular components, and various
inhibitors of ribonuclease activity (diethylpyrocarbonate, vanadyl-ribonucleoside
complex, and aurintricarboxylic acid) are described. If extremely high-quality
RNA is required (e.g., for gene expression studies), instructions are provided
for CsCl step-gradient centrifugation to remove all traces of contaminating DNA.

4: Curr Protoc Mol Biol. 2001 May;Chapter 4:Unit4.3.

Phenol/SDS method for plant RNA preparation.

[No authors listed]

The method described here can be used to prepare RNA from a variety of eukaryotic
tissues. The critical factor in isolating RNA from eukaryotic tissues is
inactivating the endogenous RNase and preventing introduction of RNase from
external sources. In general, protocols for making RNA from eukaryotic organisms
involve lysing the cells in the presence of a strong denaturant and
deproteinizing agent which inhibits RNase as well as strips the protein away from
the RNA. In this protocol, the RNA is then separated from DNA and other
impurities by selective precipitation in high salt.

5: Curr Protoc Mol Biol. 2001 May;Chapter 4:Unit4.2.

Guanidine methods for total RNA preparation.

Kingston RE, Chomczynski P, Sacchi N.

Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts,
USA.

Three different methods for RNA preparation using guanidine are presented in this
unit--a single-step isolation method employing liquid-phase separation to
selectively extract total RNA from tissues and cultured cells and two methods
that rely on a CsCl step gradient to isolate total RNA.

6: Curr Protoc Mol Biol. 2001 May;Chapter 22:Unit 22.2.

Preparation of mRNA for expression monitoring.

Byrne MC, Whitley MZ, Follettie MT.

Genetics Institute, Cambridge, Massachusetts, USA.

The ability to construct comprehensive gene expression profiles comprising
hundreds to thousands of genes whose RNA levels are monitored simultaneously
represents an exciting new capability in molecular biology. This is accomplished
by hybridizing mRNA, which has been quantitatively amplified and labeled with
biotin, to DNA chips that display thousands of nucleotides complementary to the
mRNAs of interest. In this unit, rationale for starting with poly(A(+)) vs. total
RNA is discussed, and strategies for choosing oligonucleotides for chip design is
presented. Protocols on RNA amplification and labeling, and purifying and
quantifying the cDNA and in vitro transcription products are included.